Reduced toxicity Clostridial toxin pharmaceutical compositions

ABSTRACT

A Clostridial toxin pharmaceutical composition comprising a Clostridial toxin, such as a botulinum toxin and a polysaccharide, such as a hydroxyethyl starch, wherein the pharmaceutical composition has a reduced toxicity. The Clostridial toxin pharmaceutical composition can be essentially free of any blood or serum derived proteins, filtrates or fractions.

CROSS REFERENCE

This application is a continuation in part of application Ser. No.10/288,738, filed Nov. 5, 2002 now abandoned, which is a continuation inpart of application Ser. No. 10/047,058, filed Jan. 14, 2002 nowabandoned, which is a continuation in part of application Ser. No.09/500,147, filed Feb. 8, 2000 now abandoned. The entire contents ofthese prior patent applications are incorporated herein by reference.

BACKGROUND

The present invention relates to Clostridial toxin pharmaceuticalcompositions. In particular, the present invention relates toClostridial toxin pharmaceutical compositions with a reduced toxicityand/or a reduced antigenicity and uses thereof.

A pharmaceutical composition is a formulation which contains at leastone active ingredient (such as a Clostridial toxin) as well as, forexample, one or more excipients, buffers, carriers, stabilizers,preservatives and/or bulking agents, and is suitable for administrationto a patient to achieve a desired diagnostic result or therapeuticeffect. The pharmaceutical compositions disclosed herein havediagnostic, therapeutic and/or research utility.

For storage stability and convenience of handling, a pharmaceuticalcomposition can be formulated as a lyophilized (i.e. freeze dried) orvacuum dried powder which can be reconstituted with a suitable fluid,such as saline or water, prior to administration to a patient.Alternately, the pharmaceutical composition can be formulated as anaqueous solution or suspension. A pharmaceutical composition can containa proteinaceous active ingredient. Unfortunately, a protein activeingredient can be very difficult to stabilize (i.e. maintained in astate where loss of biological activity is minimized), resultingtherefore in a loss of protein and/or loss of protein activity duringthe formulation, reconstitution (if required) and during the period ofstorage prior to use of a protein containing pharmaceutical composition.Stability problems can occur because of protein denaturation,degradation, dimerization, and/or polymerization. Various excipients,such as albumin and gelatin have been used with differing degrees ofsuccess to try and stabilize a protein active ingredient present in apharmaceutical composition. Additionally, cryoprotectants such asalcohols have been used to reduce protein denaturation under thefreezing conditions of lyophilization.

Albumin

Albumins are small, abundant plasma proteins. Human serum albumin has amolecular weight of about 69 kiloDaltons (kD) and has been used as anon-active ingredient in a pharmaceutical composition where it can serveas a bulk carrier and stabilizer of certain protein active ingredientspresent in a pharmaceutical composition.

The stabilization function of albumin in a pharmaceutical compositioncan be present both during the multi-step formulation of thepharmaceutical composition and upon the later reconstitution of theformulated pharmaceutical composition. Thus, stability can be impartedby albumin to a proteinaceous active ingredient in a pharmaceuticalcomposition by, for example, (1) reducing adhesion (commonly referred toas “stickiness”) of the protein active ingredient to surfaces, such asthe surfaces of laboratory glassware, vessels, to the vial in which thepharmaceutical composition is reconstituted and to the inside surface ofa syringe used to inject the pharmaceutical composition. Adhesion of aprotein active ingredient to surfaces can lead to loss of activeingredient and to denaturation of the remaining retained protein activeingredient, both of which reduce the total activity of the activeingredient present in the pharmaceutical composition, and; (2) reducingdenaturation of the active ingredient which can occur upon preparationof a low dilution solution of the active ingredient.

As well as being able to stabilize a protein active ingredient in apharmaceutical composition, human serum albumin also has the advantageof generally negligible immunogenicity when injected into a humanpatient. A compound with an appreciable immunogenicity can cause theproduction of antibodies against it which can lead to an anaphylacticreaction and/or to the development of drug resistance, with the diseaseor disorder to be treated thereby becoming potentially refractory to thepharmaceutical composition which has an immunogenic component.

Unfortunately, despite its known stabilizing effect, significantdrawbacks exist to the use of human serum albumin in a pharmaceuticalcomposition. For example human serum albumins are expensive andincreasingly difficult to obtain. Furthermore, blood products such asalbumin, when administered to a patient can subject the patient to apotential risk of receiving blood borne pathogens or infectious agents.Thus, it is known that the possibility exists that the presence ofalbumin in a pharmaceutical composition can result in inadvertentincorporation of infectious elements into the pharmaceuticalcomposition. For example, it has been reported that use of human serumalbumin may transmit prions into a pharmaceutical composition. A prionis a proteinaceous infectious particle which is hypothesized to arise asan abnormal conformational isoform from the same nucleic acid sequencewhich makes the normal protein. It has been further hypothesized thatinfectivity resides in a “recruitment reaction” of the normal isoformprotein to the prion protein isoform at a post translational level.Apparently the normal endogenous cellular protein is induced to misfoldinto a pathogenic prion conformation. Significantly, several lots ofhuman serum albumin have been withdrawn from distribution upon adetermination that a blood donor to a pool from which the albumin wasprepared was diagnosed with Creutzfeldt-Jacob disease.

Creutzfeldt-Jacob disease (sometimes characterized as Alzheimer'sdisease on fast forward) is a rare neurodegenerative disorder of humantransmissible spongiform encephalopathy where the transmissible agent isapparently an abnormal isoform of a prion protein. An individual withCreutzfeldt-Jacob disease can deteriorate from apparent perfect healthto akinetic mutism within six months. Possible iatrogenic transmissionof Creutzfeldt-Jacob disease by human serum albumin transfusion has beenreported and it has been speculated that sufficient protection againstCreutzfeldt-Jacob disease transmission is not provided by the usualmethods of human serum albumin preparation which methods includedisposal of blood cellular elements and heating to 60 degrees C. for 10hours. Thus, a potential risk may exist of acquiring a prion mediateddisease, such as Creutzfeldt-Jacob disease, from the administration of apharmaceutical composition which contains human plasma proteinconcentrates, such as serum albumin.

Gelatin has been used in some protein active ingredient pharmaceuticalcompositions as an albumin substitute. Notably, gelatin is a animalderived protein and therefore carries the same risk of potentialinfectivity which may be possessed by human serum albumin. Hence, it isdesirable to find a substitute for human serum albumin which is not ablood fraction, and preferably, the albumin substitute is not gelatinand is not derived from any animal (i.e. mammalian) source.

Collagen has been used cosmetically as a filler material to treat ofskin contour problems, such as to smooth smile line grooves and frownlines, folds between the eyebrows, wrinkles in the corners of the eyesand fine vertical creases above and below the lips. Collagen is alsouseful in smoothing certain post-surgical traumatic or acne scarring andviral pock marks, such as chicken pox marks. For such purposes thecollagen is injected into the dermis to raise the skin.

Botulinum Toxin

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped by morphology and function. The anaerobic, grampositive bacterium Clostridium botulinum produces a potent polypeptideneurotoxin, botulinum toxin, which causes a neuroparalytic illness inhumans and animals referred to as botulism. Clostridium botulinum andits spores are commonly found in soil and the bacterium can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of botulinum toxin (purified neurotoxincomplex) type A is a LD₅₀ in mice. Interestingly, on a molar basis,botulinum toxin type A is 1.8 billion times more lethal than diphtheria,600 million times more lethal than sodium cyanide, 30 million times morelethal than cobrotoxin and 12 million times more lethal than cholera.Singh, Critical Aspects of Bacterial Protein Toxins, pages 63-84(chapter 4) of Natural Toxins II, edited by B. R. Singh et al., PlenumPress, New York (1976) (where the stated LD₅₀ of botulinum toxin type Aof 0.3 ng equals 1 U is corrected for the fact that about 0.05 ng ofBOTOX® equals 1 unit). One unit (U) of botulinum toxin is defined as theLD₅₀ upon intraperitoneal injection into female Swiss Webster miceweighing 18-20 grams each. In other words, one unit of botulinum toxinis the amount of botulinum toxin that kills 50% of a group of femaleSwiss Webster mice. Seven generally immunologically distinct botulinumneurotoxins have been characterized, these being respectively botulinumneurotoxin serotypes A, B, C₁, D, E, F, and G, each of which isdistinguished by neutralization with type-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that botulinum toxin type Ais 500 times more potent, as measured by the rate of paralysis producedin the rat, than is botulinum toxin type B. Additionally, botulinumtoxin type B has been determined to be non-toxic in primates at a doseof 480 U/kg which is about 12 times the primate LD₅₀ for botulinum toxintype A. The botulinum toxins apparently bind with high affinity tocholinergic motor neurons, are translocated into the neuron and blockthe presynaptic release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A was approved by the U.S. Food and DrugAdministration in 1989 for the treatment of essential blepharospasm,strabismus and hemifacial spasm in patients over the age of twelve.Clinical effects of peripheral injection (i.e. intramuscular orsubcutaneous) botulinum toxin type A are usually seen within one week ofinjection, and often within a few hours after injection. The typicalduration of symptomatic relief (i.e. flaccid muscle paralysis) from asingle intramuscular injection of botulinum toxin type A can be aboutthree months to about six months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. Botulinum toxin A is a zincendopeptidase which can specifically hydrolyze a peptide linkage of theintracellular, vesicle associated protein SNAP-25. Botulinum type E alsocleaves the 25 kiloDalton (kD) synaptosomal associated protein(SNAP-25), but targets different amino acid sequences within thisprotein, as compared to botulinum toxin type A. Botulinum toxin types B,D, F and G act on vesicle-associated protein (VAMP, also calledsynaptobrevin), with each serotype cleaving the protein at a differentsite. Finally, botulinum toxin type C₁ has been shown to cleave bothsyntaxin and SNAP-25. These differences in mechanism of action mayaffect the relative potency and/or duration of action of the variousbotulinum toxin serotypes.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain (H chain) and a cell surface receptor; the receptor isthought to be different for each serotype of botulinum toxin and fortetanus toxin. The carboxyl end segment of the H chain, H_(C), appearsto be important for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and L chain. Theentire toxic activity of botulinum and tetanus toxins is contained inthe L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidasewhich selectively cleaves proteins essential for recognition and dockingof neurotransmitter-containing vesicles with the cytoplasmic surface ofthe plasma membrane, and fusion of the vesicles with the plasmamembrane. Tetanus neurotoxin, botulinum toxin B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytosolic surface of the synaptic vesicle is removed as aresult of any one of these cleavage events. Each toxin specificallycleaves a different bond.

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ are apparentlyproduced as only a 500 kD complex. Botulinum toxin type D is produced asboth 300 kD and 500 kD complexes. Finally, botulinum toxin types E and Fare produced as only approximately 300 kD complexes. The complexes (i.e.molecular weight greater than about 150 kD) are believed to contain anon-toxin hemagglutinin protein and a non-toxin and non-toxicnonhemagglutinin protein. These two non-toxin proteins (which along withthe botulinum toxin molecule can comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex. The toxincomplexes can be dissociated into toxin protein and hemagglutininproteins by treating the complex with red blood cells at pH 7.3. Thetoxin protein has a marked instability upon removal of the hemagglutininprotein.

All the botulinum toxin serotypes are made by Clostridium botulinumbacteria as inactive single chain proteins which must be cleaved ornicked by proteases to become neuroactive. The bacterial strains thatmake botulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D, and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Schantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. Raw toxin can be harvested by precipitation withsulfuric acid and concentrated by ultramicrofiltration. Purification canbe carried out by dissolving the acid precipitate in calcium chloride.The toxin can then be precipitated with cold ethanol. The precipitatecan be dissolved in sodium phosphate buffer and centrifuged. Upon dryingthere can then be obtained approximately 900 kD crystalline botulinumtoxin type A complex with a specific potency of 3×10⁷ LD₅₀ U/mg orgreater. This known process can also be used, upon separation out of thenon-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Botulinum toxins and toxin complexes can be obtained from, for example,List Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U.K.; Wako (Osaka,Japan), as well as from Sigma Chemicals of St Louis, Mo. Commerciallyavailable botulinum toxin containing pharmaceutical compositions includeBOTOX® (Botulinum toxin type A neurotoxin complex with human serumalbumin and sodium chloride) available from Allergan, Inc., of Irvine,Calif. in 100 unit vials as a lyophilized powder to be reconstitutedwith 0.9% sodium chloride before use), Dysport® (Clostridium botulinumtype A toxin haemagglutinin complex with human serum albumin and lactosein the formulation), available from Ipsen Limited, Berkshire, U.K. as apowder to be reconstituted with 0.9% sodium chloride before use), andMyoBloc™ (an injectable solution comprising botulinum toxin type B,human serum albumin, sodium succinate, and sodium chloride at about pH5.6, available from Elan Corporation, Dublin, Ireland).

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes.Additionally, pure botulinum toxin has been used to treat humans. seee.g. Kohl A., et al., Comparison of the effect of botulinum toxin A(Botox (R)) with the highly-purified neurotoxin (NT201) in the extensordigitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165. Hence, apharmaceutical composition can be prepared using a pure botulinum toxin.

The type A botulinum toxin is known to be soluble in dilute aqueoussolutions at pH 4-6.8. At pH above about 7 the stabilizing nontoxicproteins dissociate from the neurotoxin, resulting in a gradual loss oftoxicity, particularly as the pH and temperature rise. Schantz E. J., etal Preparation and characterization of botulinum toxin type A for humantreatment (in particular pages 44-45), being chapter 3 of Jankovic, J.,et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc (1994).

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin can stabilized with a stabilizingagent such as albumin and gelatin.

It has been reported that BoNt/A has been used in various clinicalsettings, including as follows:

-   -   (1) about 75-125 units of BOTOX®¹ per intramuscular injection        (multiple muscles) to treat cervical dystonia; 1Available from        Allergan, Inc., of Irvine, Ca. under the tradename BOTOX®.    -   (2) 5-10 units of BOTOX® per intramuscular injection to treat        glabellar lines (brow furrows) (5 units injected intramuscularly        into the procerus muscle and 10 units injected intramuscularly        into each corrugator supercilii muscle);    -   (3) about 30-80 units of BOTOX® to treat constipation by        intrasphincter injection of the puborectalis muscle;    -   (4) about 1-5 units per muscle of intramuscularly injected        BOTOX® to treat blepharospasm by injecting the lateral        pre-tarsal orbicularis oculi muscle of the upper lid and the        lateral pre-tarsal orbicularis oculi of the lower lid.    -   (5) to treat strabismus, extraocular muscles have been injected        intramuscularly with between about 1-5 units of BOTOX®, the        amount injected varying based upon both the size of the muscle        to be injected and the extent of muscle paralysis desired (i.e.        amount of diopter correction desired).    -   (6) to treat upper limb spasticity following stroke by        intramuscular injections of BOTOX® into five different upper        limb flexor muscles, as follows:        -   (a) flexor digitorum profundus: 7.5 U to 30 U        -   (b) flexor digitorum sublimus: 7.5 U to 30 U        -   (c) flexor carpi ulnaris: 10 U to 40 U        -   (d) flexor carpi radialis: 15 U to 60 U        -   (e) biceps brachii: 50 U to 200 U. Each of the five            indicated muscles has been injected at the same treatment            session, so that the patient receives from 90 U to 360 U of            upper limb flexor muscle BOTOX® by intramuscular injection            at each treatment session.    -   (7) to treat migraine, pericranial injected (injected        symmetrically into glabellar, frontalis and temporalis muscles)        injection of 25 U of BOTOX® has showed significant benefit as a        prophylactic treatment of migraine compared to vehicle as        measured by decreased measures of migraine frequency, maximal        severity, associated vomiting and acute medication use over the        three month period following the 25 U injection.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and insome circumstances for as long as 27 months. The Laryngoscope109:1344-1346:1999. However, the usual duration of an intramuscularinjection of Botox® is typically about 3 to 4 months.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes.Additionally, pure botulinum toxin has been used in humans. see e.g.Kohl A., et al., Comparison of the effect of botulinum toxin A (Botox(R)) with the highly-purified neurotoxin (NT 201) in the extensordigitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165 Hence, apharmaceutical composition can be prepared using a pure botulinum toxin.

The botulinum toxin molecule (about 150 kDa), as well as the botulinumtoxin complexes (about 300-900 kDa), such as the toxin type A complexare also extremely susceptible to denaturation due to surfacedenaturation, heat, and alkaline conditions. Inactivated toxin formstoxoid proteins which may be immunogenic. The resulting antibodies canrender a patient refractory to toxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) are dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin must be stabilized with astabilizing agent. To date, the only successful stabilizing agent forthis purpose has been the animal derived proteins human serum albuminand gelatin. And as indicated, the presence of animal derived proteinsin the final formulation presents potential problems in that certainstable viruses, prions, or other infectious or pathogenic compoundscarried through from donors can contaminate the toxin.

Furthermore, any one of the harsh pH, temperature and concentrationrange conditions required to lyophilize (freeze-dry) or vacuum dry abotulinum toxin containing pharmaceutical composition into a toxinshipping and storage format (ready for use or reconstitution by aphysician) can detoxify the toxin. Thus, animal derived or donor poolproteins such as gelatin and serum albumin have been used with somesuccess to stabilize botulinum toxin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, human serum albumin, and sodium chloride packaged insterile, vacuum-dried form. The botulinum toxin type A is made from aculture of the Hall strain of Clostridium botulinum grown in a mediumcontaining N-Z amine and yeast extract. The botulinum toxin type Acomplex is purified from the culture solution by a series of acidprecipitations to a crystalline complex consisting of the active highmolecular weight toxin protein and an associated hemagglutinin protein.The crystalline complex is re-dissolved in a solution containing salineand albumin and sterile filtered (0.2 microns) prior to vacuum-drying.BOTOX® can be reconstituted with sterile, non-preserved saline prior tointramuscular injection. Each vial of BOTOX® contains about 100 units(U) of Clostridium botulinum toxin type A complex, 0.5 milligrams ofhuman serum albumin and 0.9 milligrams of sodium chloride in a sterile,vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX® sterile normal saline without apreservative (0.9% Sodium Chloride injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is denatured by bubbling or similar violent agitation, the diluent isgently injected into the vial. For sterility reasons, BOTOX® should beadministered within four hours after reconstitution. During this timeperiod, reconstituted BOTOX® is stored in a refrigerator (2° to 8° C.).Reconstituted BOTOX® is clear, colorless and free of particulate matter.The vacuum-dried product is stored in a freezer at or below −5° C.

It has been reported that a suitable alternative to human serum toalbumin as a botulinum toxin stabilizer may be another protein oralternatively a low molecular weight (non-protein) compound. Carpenderet al., Interactions of Stabilizing Additives with Proteins DuringFreeze-Thawing and Freeze-Drying, International Symposium on BiologicalProduct Freeze-Drying and Formulation, 24-26 Oct. 1990; Karger (1992),225-239.

Many substances commonly used as carriers and bulking agents inpharmaceutical compositions have proven to be unsuitable as albuminreplacements in a Clostridial toxin containing pharmaceuticalcomposition. For example, the disaccharide cellobiose has been found tobe unsuitable as a botulinum toxin stabilizer. Thus, it is known thatthe use of cellobiose as an excipient in conjunction with albumin andsodium chloride results in a much lower level of toxicity (10% recovery)after lyophilization of crystalline botulinum toxin type A with theseexcipients, as compared to the toxicity after lyophilization with onlyhuman serum albumin (>75% to >90% recovery). Goodnough et al.,Stabilization of Botulinum Toxin Type A During Lyophilization, App &Envir. Micro. 58 (10) 3426-3428 (1992).

Furthermore, saccharides, including polysaccharides, are in general poorcandidates to serve as protein stabilizers. Thus, it is known that apharmaceutical composition containing a protein active ingredient isinherently unstable if the protein formulation comprises a saccharide(such as glucose or a polymer of glucose) or carbohydrates becauseproteins and glucose are known to interact together and to undergo thewell-described Maillard reaction, due to the reducing nature of glucoseand glucose polymers. Much work has been dedicated to mostlyunsuccessful attempts at preventing this protein-saccharide reaction by,for example, reduction of moisture or use of non-reducing sugars.Significantly, the degradative pathway of the Maillard reaction canresult in a therapeutic insufficiency of the protein active ingredient.A pharmaceutical formulation comprising protein and a reducingsaccharide, carbohydrate or sugar, such as a glucose polymer, istherefore inherently unstable and cannot be stored for a long period oftime without significant loss of the active ingredient protein's desiredbiological activity.

Notably, one of the reasons human serum albumin can function effectivelyas a stabilizer of a protein active ingredient in a pharmaceuticalcomposition is because, albumin, being a protein, does not undergo theMaillard reaction with the protein active ingredient in a pharmaceuticalcomposition. Hence, one would expect to find and to look for asubstitute for human serum albumin amongst other proteins.

Finding an appropriate substitute for human serum albumin as astabilizer of the botulinum toxin present in a pharmaceuticalcomposition is difficult and problematic because human serum albumin isbelieved to function in a pharmaceutical composition as more than a merebulking agent. Thus, albumin apparently can interact with botulinumtoxin so as to increase the potency of the neurotoxin. For example, itis known that bovine serum albumin can act as more than a merestabilizing excipient for botulinum toxin type A, since bovine serumalbumin apparently also accelerates the rate of catalysis of syntheticpeptide substrates, which substrates resemble the SNAP-25 intraneuronalsubstrate for botulinum toxin type A Schmidt, et al., EndoproteinaseActivity of Type A Botulinum Neurotoxin Substrate Requirements andActivation by Serum Albumin, J. of Protein Chemistry, 16 (1), 19-26(1997). Thus, albumin may have a potentiating effect, apparently byaffecting rate kinetics, upon the intracellular proteolytic action of abotulinum toxin upon the toxin's substrate. This potentiating effect maybe due to albumin which has accompanied the botulinum toxin uponendocytosis of the toxin into a target neuron or the potentiating effectmay be due to the pre-existing presence cytoplasmic albumin within theneuron protein prior to endocytosis of the botulinum toxin.

The discovery of the presence of a kinetic rate stimulatory effect bybovine serum albumin upon the proteolytic activity of botulinum toxintype A renders the search for a suitable substitute for albumin in abotulinum toxin containing pharmaceutical formulation especiallyproblematic. Thus, an albumin substitute with desirable toxinstabilization characteristics may have an unknown and possiblydeleterious effect upon the rate of substrate catalysis by the toxin,since at least with regard to bovine serum albumin the twocharacteristics (toxin stabilization and toxin substrate catalysispotentiation) are apparently inherent to the same albumin excipient.This potentiating effect of albumin shows that albumin does not act as amere excipient in the formulation and therefore renders the search for asuitable substitute for albumin more difficult.

Additionally there are many unique characteristics of botulinum toxinand its formulation into a suitable pharmaceutical composition whichconstrain and hinder and render the search for a replacement for thealbumin used in current botulinum toxin containing pharmaceuticalformulations very problematic. Examples of four of these uniquecharacteristics follow.

First, botulinum toxin is a relatively large protein for incorporationinto a pharmaceutical formulation (the molecular weight of the botulinumtoxin type A complex is 900 kD) and is therefore is inherently fragileand labile. The size of the toxin complex makes it much more friable andlabile than smaller, less complex proteins, thereby compounding theformulation and handling difficulties if toxin stability is to bemaintained. Hence, an albumin replacement must be able to interact withthe toxin in a manner which does not denature, fragment or otherwisedetoxify the toxin molecule or cause disassociation of the non-toxinproteins present in the toxin complex.

Second, as the most lethal known biological product, exceptional safety,precision, and accuracy is called for at all steps of the formulation ofa botulinum toxin containing pharmaceutical composition. Thus, apreferred potential albumin replacer should not itself be toxic ordifficult to handle so as to not exacerbate the already extremelystringent botulinum toxin containing pharmaceutical compositionformulation requirements.

Third, since botulinum toxin was the first microbial toxin to beapproved (by the FDA in 1989) for injection for the treatment of humandisease, specific protocols had to be developed and approved for theculturing, bulk production, formulation into a pharmaceutical and use ofbotulinum toxin. Important considerations are toxin purity and dose forinjection. The production by culturing and the purification must becarried out so that the toxin is not exposed to any substance that mightcontaminate the final product in even trace amounts and cause unduereactions in the patient. These restrictions require culturing insimplified medium without the use of animal meat products andpurification by procedures not involving synthetic solvents or resins.Preparation of toxin using enzymes, various exchangers, such as thosepresent in chromatography columns and synthetic solvents can introducecontaminants and are therefore excluded from preferred formulationsteps. Furthermore, botulinum toxin type A is readily denatured attemperatures above 40 degrees C., loses toxicity when bubbles form atthe air/liquid interface, and denatures in the presence of nitrogen orcarbon dioxide.

Fourth, particular difficulties exist to stabilize botulinum toxin typeA, because type A consists of a toxin molecule of about 150 kD innoncovalent association with nontoxin proteins weighing about 750 kD.The nontoxin proteins are believed to preserve or help stabilize thesecondary and tertiary structures upon which toxicity is dependant.Procedures or protocols applicable to the stabilization of nonproteinsor to relatively smaller proteins are not applicable to the problemsinherent with stabilization of the botulinum toxin complexes, such asthe 900 kD botulinum toxin type A complex. Thus while from pH 3.5 to 6.8the type A toxin and non toxin proteins are bound togethernoncovalently, under slightly alkaline conditions (pH>7.1) the verylabile toxin is released from the toxin complex. As set forthpreviously, pure botulinum toxin (i.e. the 150 kD molecule) has beenproposed as the active ingredient in a pharmaceutical composition.

In light of the unique nature of botulinum toxin and the requirementsset forth above, the probability of finding a suitable albuminreplacement for the human serum albumin used in current botulinum toxincontaining pharmaceutical compositions must realistically be seen toapproach zero. Prior to the present invention, only the animal derivedproteins, human serum albumin and gelatin, had been known to haveutility as suitable stabilizers of the botulinum toxin present in apharmaceutical formulation. Thus, albumin, by itself or with one or moreadditional substances such as sodium phosphate or sodium citrate, isknown to permit high recovery of toxicity of botulinum toxin type Aafter lyophilization. Unfortunately, as already set forth, human serumalbumin, as a pooled blood product, can, at least potentially, carryinfectious or disease causing elements when present in a pharmaceuticalcomposition. Indeed, any animal product or protein such as human serumalbumin or gelatin can also potentially contain pyrogens or othersubstances that can cause adverse reactions upon injection into apatient.

Chinese patent application CN 1215084A discusses an albumin freebotulinum toxin type A formulated with gelatin, an animal derivedprotein. U.S. Pat. No. 6,087,327 also discloses a composition ofbotulinum toxin types A and B formulated with gelatin. Theseformulations therefore do not eliminate the risk of transmitting ananimal protein derived or accompanying infectious element.

Tetanus toxin, as wells as derivatives (i.e. with a non-native targetingmoiety), fragments, hybrids and chimeras thereof can also havetherapeutic utility. The tetanus toxin bears many similarities to thebotulinum toxins. Thus, both the tetanus toxin and the botulinum toxinsare polypeptides made by closely related species of Clostridium(Clostridium tetani and Clostridium botulinum, respectively).Additionally, both the tetanus toxin and the botulinum toxins aredichain proteins composed of a light chain (molecular weight about 50kD) covalently bound by a single disulfide bond to a heavy chain(molecular weight about 100 kD). Hence, the molecular weight of tetanustoxin and of each of the seven botulinum toxins (non-complexed) is about150 kD. Furthermore, for both the tetanus toxin and the botulinumtoxins, the light chain bears the domain which exhibits intracellularbiological (protease) activity, while the heavy chain comprises thereceptor binding (immunogenic) and cell membrane translocationaldomains.

Further, both the tetanus toxin and the botulinum toxins exhibit a high,specific affinity for gangliocide receptors on the surface ofpresynaptic cholinergic neurons. Receptor mediated endocytosis oftetanus toxin by peripheral cholinergic neurons results in retrogradeaxonal transport, blocking of the release of inhibitoryneurotransmitters from central synapses and a spastic paralysis.Contrarily, receptor mediated endocytosis of botulinum toxin byperipheral cholinergic neurons results in little if any retrogradetransport, inhibition of acetylcholine exocytosis from the intoxicatedperipheral motor neurons and a flaccid paralysis.

Finally, the tetanus toxin and the botulinum toxins resemble each otherin both biosynthesis and molecular architecture. Thus, there is anoverall 34% identity between the protein sequences of tetanus toxin andbotulinum toxin type A, and a sequence identity as high as 62% for somefunctional domains. Binz T. et al., The Complete Sequence of BotulinumNeurotoxin Type A and Comparison with Other Clostridial Neurotoxins, JBiological Chemistry 265(16); 9153-9158:1990.

As set forth above, foodborne botulism can result from swallowingbotulinum toxin present in improperly preserved food. The toxin can passunattenuated across gut mucosa into the general circulation and iscarried by the bloodstream to peripheral cholinergic synapses where itblocks acetylcholine release causes impaired autonomic and neuromusculartransmission, one of the primary symptoms being flaccid muscleparalysis. Similarly ingestion or injection of tetanus toxin can resultin the spastic paralysis characteristic of tetanus. As expected,intravenous administration of botulinum toxin causes botulism and can befatal depending upon factors such as the dose of toxin administered andthe condition of the patient. See e.g. Arnon, S. S., Clinical Botulism,chapter 13, pages 145-150 of Brin M. F. et al, editors, Scientific andtherapeutic aspects of botulinum Toxin, Lippincott, Williams & Wilkins(2002), and; Kondo H., et al, Titration of botulinum toxins for lethaltoxicity by intravenous injection into mice, Jpn J Med Sci Biol 1984;37:131-5.

Clinical use of botulinum toxin is typically by subcutaneous orintramuscular administration in order to treat, for example, a spasticmuscle disorder or for the cosmetic alleviation of hyperkinetic facialwrinkles. Through errors or incompetence in physician administrationtechnique, inadvertent overdosing and/or patient sensitivity to thetoxin, iatrogenic intoxication, as revealed symptomatically bywidespread muscle weakness, can occur.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephrine. In most instances acetylcholine has an excitatoryeffect. However, acetylcholine is known to have inhibitory effects atsome of the peripheral parasympathetic nerve endings, such as inhibitionof heart rate by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic, neurons of the parasympathetic nervoussystem as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the adrenal medulla, as well as within the autonomic ganglia,that is on the cell surface of the postganglionic neuron at the synapsebetween the preganglionic and postganglionic neurons of both thesympathetic and parasympathetic systems. Nicotinic receptors are alsofound in many nonautonomic nerve endings, for example in the membranesof skeletal muscle fibers at the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and parathyroid hormone, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

A neuromuscular junction is formed in skeletal muscle by the proximityof axons to muscle cells. A signal transmitted through the nervoussystem results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscular junction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

Hydroxyethyl Starch

A polysaccharide can be made up of hundreds or even thousands ofmonosaccharide units held together by glycoside (ether) linkages. Twoimportant polysaccharides are cellulose and starch. Cellulose is thechief structural material in plants, giving plants their rigidity andform. Starch makes up the reserve food supply of plants and is foundmainly in various seeds and tubers.

Starch occurs as granules whose size and shape are characteristic of theplant from which the starch is obtained. In general about 80% of starchis a water insoluble fraction called amylopectin. Amylopectin is made upof chains of D-glucose (as glucopyranose) units, each unit being joinedby an alpha glycoside linkage to C-4 of the next glucose unit. Likestarch, cellulose is also made up of chains of D-glucose units, whereeach unit is joined by a glucoside linkage to the C-4 of the next unit.Unlike starch though, the glycoside linkages in cellulose are betalinkages. Treatment of cellulose with sulfuric acid and acetic anhydrideyields the disaccharide cellobiose. As previously set forth, attempts tostabilize botulinum toxin using cellobiose have been unsuccessful.

A particular starch derivative which can be obtained by treating starchwith pyridine and ethylene chlorohydrin, is 2-hydroxyethyl starch, alsocalled hetastarch. U.S. Pat. No. 4,457,916 discloses a combination of anonionic surfactant and hydroxyethyl starch to stabilize aqueoussolutions of tumor necrosis factor (TNF). Additionally, a 6% aqueoussolution of 2-hydroxyethyl starch (hetastarch) (available from DupontPharma, Wilmington, Del. under the trade name HESPAN®, 6% hetastarch in0.9% sodium chloride injection) is known. Albumin is known to act as aplasma volume expander upon intravenous administration to a patient.HESPAN® has also been administrated to patients to achieve a plasmavolume expansion effect and in that sense intravenous HESPAN® can beconsidered a replacement for intravenous albumin.

Hetastarch is an artificial colloid derived from a waxy starch composedalmost entirely of amylopectin. Hetastarch can be obtained byintroducing hydroxyethyl ether groups onto glucose units of the starch,and the resultant material can then be hydrolyzed to yield a productwith a molecular weight suitable for use as a plasma volume expander.Hetastarch is characterized by its molar substitution and also by itsmolecular weight. The molar substitution can be approximately 0.75,meaning that hetastarch is etherified to the extent that for every 100glucose units of hetastarch there are, on average, approximately 75hydroxyethyl substituent groups. The average molecular weight ofhetastarch is approximately 670 kD with a range of 450 kD to 800 kD andwith at least 80% of the polymer units falling within the range of 20 kDto 2,500 kD. Hydroxyethyl groups are attached by ether linkagesprimarily at C-2 of the glucose unit and to a lesser extent at C-3 andC-6. The polymer resembles glycogen, and the polymerized D-glucose unitsare joined primarily by α-1,4 linkages with occasional α-1,6 branchinglinkages. The degree of branching is approximately 1:20, meaning thatthere is an average of approximately one α-1,6 branch for every 20glucose monomer units. Hetastarch is comprised of more than 90%amylopectin.

The plasma volume expansion produced by HESPAN® can approximate thatobtained with albumin. Hetastarch molecules below 50 kD molecular weightare rapidly eliminated by renal excretion and a single dose ofapproximately 500 mL of HESPAN® (approximately 30 g) results inelimination in the urine of approximately 33% of the administeredHESPAN® within about 24 hours. The hydroxyethyl group of hydroxyethylstarch is not cleaved in vivo, but remains intact and attached toglucose units when excreted. Significant quantities of glucose are notproduced as hydroxyethylation prevents complete metabolism of thesmaller hydroxyethyl starch polymers

Cellulose can likewise be converted to a hydroxyethyl cellulose. Theaverage molecular weight of 2-hydroxyethyl cellulose (a 2-hydroxyethylether of cellulose) is about 90 kD. Unfortunately, hydroxyethylcellulose, unlike hydroxyethyl starch, is highly reactive and thereforeunsuited for use as a stabilizer of a protein active ingredient in apharmaceutical formulation.

What is needed therefore is a botulinum toxin containing pharmaceuticalcomposition which is free of animal derived proteins such as a bloodpooled or blood fraction derived serum albumin or gelatin and which hasa reduced toxicity and/or a reduced antigenicity.

SUMMARY

The present invention meets this need and provides a botulinum toxinpharmaceutical composition which is free of animal derived proteins suchas a blood pooled or blood fraction derived human serum albumin orgelatin, and which has a reduced toxicity and/or a reducedimmunogenicity. One embodiment of the present invention is a botulinumtoxin pharmaceutical composition wherein the primary stabilizer presentin the formulation is a polysaccharide.

The present invention encompasses new pharmaceutical compositions,embodiments of which can provide a superior replacement (i.e. by use ofa suitable polysaccharide, with or without additional stabilizers) forthe serum or native albumin present as a primary stabilizer in apharmaceutical composition. Thus, my invention encompasses new,stabilized botulinum toxin pharmaceutical compositions, which can havethe additional and desirable characteristic of reduced toxicity, such asa reduced systemic toxicity (i.e. upon intravenous administration) orsuch as a reduced toxicity upon intramuscular administration. The serumor native albumin replacement compound used has the characteristic oflow, and preferably negligible, immunogenicity when injected into apatient.

DEFINITIONS

As used herein, the words or terms set forth below have the followingdefinitions.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Administration”, or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject. Thepharmaceutical compositions disclosed herein are “locally administered”by e.g. intramuscular (i.m.), intradermal, subcutaneous administration,intrathecal administration, intraperitoneal (i.p.) administration,topical (transdermal) and implantation (i.e. of a slow-release devicesuch as polymeric implant or miniosmotic pump) routes of administration.

“Amino acid’ includes polyamino acids.

“Animal protein free” means the absence of blood derived, blood pooledand other animal derived products or compounds. “Animal” means a mammal(such as a human), bird, reptile, fish, insect, spider or other animalspecies. “Animal” excludes microorganisms, such as bacteria. Thus, ananimal protein free pharmaceutical composition within the scope of myinvention can include a Clostridial neurotoxin. For example, an animalprotein free pharmaceutical composition means a pharmaceuticalcomposition which is either substantially free or essentially free orentirely free of a serum derived albumin, gelatin and other animalderived proteins, such as immunoglobulins. An example of an animalprotein free pharmaceutical composition is a pharmaceutical compositionwhich comprises or which consists of a botulinum toxin (as the activeingredient) and a suitable polysaccharide as a stabilizer or excipient.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum,as well as a botulinum toxin (or the light chain or the heavy chainthereof) made recombinantly by a non-Clostridial species. The phrase“botulinum toxin”, as used herein, encompasses the botulinum toxinserotypes A, B, C, D, E, F and G. Botulinum toxin, as used herein, alsoencompasses both a botulinum toxin complex (i.e. the 300, 600 and 900kDa complexes) as well as the purified botulinum toxin (i.e. about 150kDa). “Purified botulinum toxin” is defined as a botulinum toxin that isisolated, or substantially isolated, from other proteins, includingproteins that form a botulinum toxin complex. A purified botulinum toxinmay be greater than 95% pure, and preferably is greater than 99% pure.The botulinum C₂ and C₃ cytotoxins, not being neurotoxins, are excludedfrom the scope of the present invention.

“Clostridial neurotoxin” means a neurotoxin produced from, or native to,a Clostridial bacterium, such as Clostridium botulinum, Clostridiumbutyricum or Clostridium beratti, as well as a Clostridial neurotoxinmade recombinantly by a non-Clostridial species.

“Entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

“Essentially free” (or “consisting essentially of”’) means that onlytrace amounts of the substance can be detected.

“Immobilizing” means a step that prevents a subject from moving one ormore body parts. If a sufficient number of body parts are immobilized,the subject will accordingly be immobilized. Thus, “immobilizing”encompasses the immobilization of a body part, such as a limb, and/orthe complete immobilization of a subject.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Patient” means a human or non-human subject receiving medical orveterinary care. Accordingly, as disclosed herein, the compositions maybe used in treating any animal, such as mammals.

“Pharmaceutical composition” means a formulation in which an activeingredient can be a neurotoxin, such as a Clostridial neurotoxin. Theword “formulation” means that there is at least one additionalingredient in the pharmaceutical composition besides a neurotoxin activeingredient. A pharmaceutical composition is therefore a formulationwhich is suitable for diagnostic or therapeutic administration (i.e. byintramuscular or subcutaneous injection or by insertion of a depot orimplant) to a subject, such as a human patient. The pharmaceuticalcomposition can be: in a lyophilized or vacuum dried condition; asolution formed after reconstitution of the lyophilized or vacuum driedpharmaceutical composition with saline or water, or; as a solution whichdoes not require reconstitution. The neurotoxin active ingredient can beone of the botulinum toxin serotypes A, B, C₁, D, E, F or G or a tetanustoxin, all of which can be made natively by Clostridial bacteria. Asstated, a pharmaceutical composition can be liquid or solid, for examplevacuum-dried. The constituent ingredients of a pharmaceuticalcomposition can be included in a single composition (that is all theconstituent ingredients, except for any required reconstitution fluid,are present at the time of initial compounding of the pharmaceuticalcomposition) or as a two-component system, for example a vacuum-driedcomposition reconstituted with a diluent such as saline which diluentcontains an ingredient not present in the initial compounding of thepharmaceutical composition. A two-component system provides the benefitof allowing incorporation of ingredients which are not sufficientlycompatible for long-term shelf storage with the first component of thetwo component system. For example, the reconstitution vehicle or diluentmay include a preservative which provides sufficient protection againstmicrobial growth for the use period, for example one-week ofrefrigerated storage, but is not present during the two-year freezerstorage period during which time it might degrade the toxin. Otheringredients, which may not be compatible with a Clostridial toxin orother ingredients for long periods of time, may be incorporated in thismanner; that is, added in a second vehicle (i.e. in the reconstitutionfluid) at the approximate time of use.

“Polysaccharide” means a polymer of more than two saccharide moleculemonomers, which monomers can be identical or different.

“Protein stabilizer” (or “primary stabilizer”) is a chemical agent thatassists to preserve or maintain the biological structure (i.e. the threedimensional conformation) and/or biological activity of a protein (suchas a Clostridial neurotoxin, such as a botulinum toxin). Stabilizers canbe proteins or polysaccharides. Examples of protein stabilizers includehydroxyethyl starch (hetastarch), serum albumin, gelatin, collagen, aswell as a recombinant albumin, gelatin or collagen. As disclosed herein,the primary stabilizer can be a synthetic agent that would not producean immunogenic response (or produces an attenuated immune response) in asubject receiving a composition containing the primary stabilizer. Inother embodiments of the invention, the protein stabilizers may beproteins from the same species of animal that is being administered theprotein. Additional stabilizers may also be included in a pharmaceuticalcomposition. These additional or secondary stabilizers may be used aloneor in combination with primary stabilizers, such as proteins andpolysaccharides. Exemplary secondary stabilizers include, but are notlimited to non-oxidizing amino acid derivatives (such as a tryptophanderivate, such as N-acetyl-tryptophan (“NAT”)), caprylate (i.e. sodiumcaprylate), a polysorbate (i.e. P80), amino acids, and divalent metalcations such as zinc. A pharmaceutical composition can also includepreservative agents such as benzyl alcohol, benzoic acid, phenol,parabens and sorbic acid. A “recombinant stabilizer” is a “primarystabilizer” made by recombinant means, such as for example, arecombinantly made albumin (such as a recombinantly made human serumalbumin), collagen, gelatin or a cresol, such as an M-cresol.

“Reduced toxicity” means, with regard to a first pharmaceuticalcomposition (i.e. Formulation II or IIA) with a particular proteinactive ingredient (i.e. a botulinum toxin) and a particular excipient orprotein stabilizer, that the first pharmaceutical composition can beadministered (i.e. by an intramuscular or intravenous route ofadministration) to a mammal at a dose level which is at the same as oreven twice what is a fatal dose of a second pharmaceutical composition(i.e. Formulation I) administered by the same route, which secondpharmaceutical composition has the same protein active ingredient (i.e.a botulinum toxin) but which does not have the same particular excipientor protein stabilizer, without death resulting to the mammal to whichthe first pharmaceutical composition is administered.

“Stabilizing”, “stabilizes”, or “stabilization” mean that apharmaceutical active ingredient (“PAI”) retains at least 20% and up to100% of its biological activity (which can be assessed as potency or astoxicity by an in vivo LD₅₀ or ED₅₀ measure) in the presence of acompound which is stabilizing, stabilizes or which providesstabilization to the PAI. For example, upon (1) preparation of serialdilutions from a bulk or stock solution, or (2) upon reconstitution withsaline or water of a lyophilized, or vacuum dried botulinum toxincontaining pharmaceutical composition which has been stored at or belowabout −2 degrees C. for between six months and four years, or (3) for anaqueous solution botulinum toxin containing pharmaceutical compositionwhich has been stored at between about 2 degrees and about 8 degrees C.for from six months to four years, the botulinum toxin present in thereconstituted or aqueous solution pharmaceutical composition has (in thepresence of a compound which is stabilizing, stabilizes or whichprovides stabilization to the PAI) greater than about 20% and up toabout 100% of the potency or toxicity that the biologically activebotulinum toxin had prior to being incorporated into the pharmaceuticalcomposition.

“Substantially free” means present at a level of less than one percentby weight of the pharmaceutical composition.

“Therapeutic formulation” means a formulation can be used to treat andthereby alleviate a disorder or a disease, such as a disorder or adisease characterized by hyperactivity (i.e. spasticity) of a peripheralmuscle.

A pharmaceutical composition within the scope of my invention cancomprise a Clostridial toxin, such as a botulinum toxin, and apolysaccharide as a primary stabilizer (as a protein stabilizer), andhave a reduced toxicity.

The botulinum toxin can be present as a botulinum toxin complex (i.e. asan approximately 300 to about 900 kiloDalton complex depending upon theparticular botulinum toxin serotype) or the botulinum toxin can be ispresent as a pure or purified botulinum toxin (i.e. as the botulinumtoxin molecule of about 150 kiloDaltons). The pharmaceutical compositioncan also comprise a secondary stabilizer, such as a metal (i.e. zinc) orNAT.

Significantly, a pharmaceutical composition within the scope of myinvention can have an enhanced potency or stability. By enhanced potencyit is meant that the potency of a first botulinum toxin pharmaceuticalcomposition is greater than the potency of a second botulinum toxinpharmaceutical composition.

Furthermore, a pharmaceutical composition within the scope of myinvention can have an enhanced anti-microbial activity. By enhancedanti-microbial activity it is meant that the ability of a firstbotulinum toxin pharmaceutical composition with a particular componentto inhibit the growth in a liquid solution of a particular microorganismis greater than the ability of a liquid solution of a second botulinumtoxin pharmaceutical composition without the particular componentpresent in the first botulinum toxin pharmaceutical composition toinhibit the growth of the same microorganism under the same conditions.

Another preferred embodiment of my invention is a pharmaceuticalcomposition which can comprise (or which can consist essentially of orwhich can consist of) a botulinum toxin, a primary stabilizer, and asecondary stabilizer.

A pharmaceutical composition within the scope of the present inventioncan also include a neurotoxin, and a polysaccharide and have a reducedtoxicity. The polysaccharide stabilizes the neurotoxin. Thepharmaceutical compositions disclosed herein can have a pH of betweenabout 5 and 7.3 when reconstituted or upon injection. The averagemolecular weight of a disaccharide unit of the polysaccharide ispreferably between about 345 D and about 2,000 D. In a more preferredembodiment, the average molecular weight of a disaccharide unit of thepolysaccharide is between about 350 kD and about 1,000 kD and in a mostpreferred embodiment between about 375 D and about 700 D. Additionally,the polysaccharide can comprise at least about 70% amylopectin.Furthermore, the weight average molecular weight of the polysaccharideitself is between about 20 kD and about 2,500 kD.

Preferably, substantially all of the disaccharide units of thepolysaccharide comprise ether linked glucopyranose molecules. An averageof about 4 to about 10 of the hydroxyl groups present on each 10 of theglucopyranoses present in the polysaccharide are substituted, through anether linkage, with a compound of the formula (CH₂)_(n)—OH, where n canbe an integer from 1 to 10. More preferably, n is an integer between 1and 3.

In a particularly preferred embodiment, an average of about 6 to about 9of the hydroxyl groups present on each 10 of the glucopyranoses presentin the polysaccharide are substituted, through an ether linkage, with acompound of the formula (CH₂)_(n)—OH, where n can be an integer from 1to 10. And in a most preferred embodiment, an average of about 7 toabout 8 of the hydroxyl groups present on each 10 of the glucopyranosespresent in the polysaccharide are substituted, through an ether linkage,with a compound of the formula (CH₂)_(n)—OH, where n can be an integerfrom 1 to 10.

A detailed embodiment of the present invention can be a pharmaceuticalcomposition with a reduced toxicity and suitable for injection into ahuman patient, which includes a botulinum toxin, and a polysaccharide.The polysaccharide can comprise a plurality of linked glucopyranoseunits, each glucopyranose unit having a plurality of hydroxyl groups,wherein an average of about 6 to about 9 of the hydroxyl groups presenton each 10 of the glucopyranoses present in the polysaccharide aresubstituted, through an ether linkage, with a compound of the formula(CH₂)_(n)—OH, where n can be an integer from 1 to 4. The polysaccharidecan be an ethyl ether substituted polysaccharide.

The pharmaceutical composition is suitable for administration to a humanpatent to achieve a therapeutic effect, and the neurotoxin can be one ofthe botulinum toxin serotypes A, B, C₁, D, E, F and G. In a preferredembodiment of the present invention, the pharmaceutical compositioncomprises a botulinum toxin, and a hydroxyethyl starch.

Another embodiment of the present invention can encompass apharmaceutical composition which includes a botulinum toxin, apolysaccharide, and an amino acid or a polyamino acid.

Whether the pharmaceutical composition comprises, beside the neurotoxinactive ingredient, only a polysaccharide stabilizer, only an amino acidstabilizer or both polysaccharide and amino acid stabilizers, thepharmaceutical composition retains its potency substantially unchangedfor six month, one year, two year, three year and/or four year periodswhen stored at a temperature between about −1° C. and about −15° C.Additionally, the indicated pharmaceutical compositions can have apotency or % recovery of between about 20% and about 100% uponreconstitution. Alternately or in addition, the pharmaceuticalcomposition can have a potency of between about 10 U/mg and about 30U/mg upon reconstitution, such as a potency of about 20 U/mg uponreconstitution. Significantly, the pharmaceutical composition is devoidof any albumin. Thus, the pharmaceutical composition can besubstantially free of any non-toxin complex proteins. Notably, the aminoacid can be present in an amount of between about 0.5 mg and about 1.5mg of amino acid per 100 units of botulinum toxin.

The polysaccharide can be a starch such as a hydroxyethyl starch, which,when the pharmaceutical composition comprises about 100 units of thebotulinum toxin, there can be between about 500 μg and about 700 μg ofthe hydroxyethyl starch present. Preferably, the botulinum toxin is isbotulinum toxin type A, and the amino acid, when present, is selectedfrom the group consisting of lysine, glycine, histidine and arginine.

A further detailed embodiment of the present invention can be a stable,high potency, non-pyrogenic, vacuum dried botulinum toxin type Apharmaceutical composition, comprising a botulinum toxin type A complex,a polysaccharide, and, an amino acid or a polyamino acid. Thepharmaceutical composition can be albumin free, have a 1 year shelf lifeat −5 C. with about 90% potency immediately upon reconstitution withsaline or water and about 80% potency 72 hours after reconstitution andstorage at 2 C. Additionally, the pharmaceutical composition can have aspecific toxicity of at least about 10⁷ U/mg upon reconstitution.

My invention also encompasses a botulinum toxin formulation whichcomprises a botulinum toxin hydroxyethyl starch (HES) glycine andprovidine.

The present invention also encompasses a lyophilized or vacuum driedpharmaceutical composition consisting essentially of a high molecularweight polysaccharide and a botulinum toxin, wherein the botulinum toxinis stabilized by the high molecular weight polysaccharide. The highmolecule weight polysaccharide can be selected from the group consistingof hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch,hydroxybutyl starch, and hydroxypentyl starch and The botulinum toxincan be selected from the group consisting of botulinum toxin types A, B,C₁, D, E, F and G.

The polysaccharide can be present in the pharmaceutical composition inan amount of between about 1×10⁻⁹ moles of the polysaccharide per unitof a botulinum toxin to about 2×10⁻¹² moles of the polysaccharide perunit of the botulinum toxin.

The present invention also includes (a) a method for making apharmaceutical composition, comprising the step of preparing a mixtureof a botulinum toxin and a polysaccharide comprised of covalently linkedrepeating monomers, wherein the average monomer molecular weight isbetween about 350 D and about 1,000 D, and (b) a method for stabilizinga clostridial neurotoxin against denaturation or aggregation, the methodcomprising the step of contacting a clostridial neurotoxin with astabilizing composition comprising a polysaccharide. The contacting stepin this later method can comprise the step of adding to an aqueoussolution or to a lyophilized or vacuum dried powder containing aclostridial neurotoxin an effective amount of the polysaccharide.

A further aspect of the present invention is a pharmaceuticalcomposition, comprising a botulinum toxin, and a recombinantly madealbumin. This composition preferably also includes anacetyltryptophanate and salts and derivatives thereof.

An additional embodiment of the present invention is a device forinjecting a pharmaceutical composition comprising a dual chamberprefilled syringe, one chamber of which syringe contains a botulinumtoxin and the second chamber of which syringe contains a diluent orbuffer.

A further embodiment of the present invention is a method for using apharmaceutical composition, the method comprising the step of localadministration of the pharmaceutical composition to a patient to achievea therapeutic effect, wherein the pharmaceutical composition comprises abotulinum toxin, a polysaccharide and an amino acid.

Significantly, my invention also includes a pharmaceutical composition,comprising a botulinum toxin, and an amino acid. My invention alsoincludes a stable pharmaceutical composition, consisting essentially ofa botulinum toxin, and an amino acid. These pharmaceutical compositionscan be albumin free, polysaccharide free, has a 1 year shelf life at −5C. with at least about 90% potency upon reconstitution with saline orwater and about 80% potency 72 hours after reconstitution and storage at2 C.

In one aspect of the invention, a method for immobilizing a mammalcomprises the step of administering a composition, which comprises atleast one botulinum toxin serotype and a polysaccharide that stabilizesthe botulinum toxin and is nonimmunogenic to the mammal. In oneembodiment, the foregoing method may be practiced by administering acomposition comprising a hetastarch.

In another embodiment of the invention, a method for immobilizing amammal, comprises the step of administering a composition to the mammal,wherein the composition comprises (i) at least one botulinum toxinserotype, and (ii) a polysaccharide, which comprises a plurality oflinked glucopyranose units that each have a plurality of hydroxyl groupspresent on each of the glucopyranoses present in the polysaccharide aresubstituted, through an ether linkage, with a compound of the formula(CH₂)_(n)—OH, where n can be an integer from 1 to 4.

In another embodiment of the invention, a method for immobilizing amammal comprises the step of administering a composition to the mammal,wherein the composition comprises a botulinum toxin, and a hydroxyethylstarch, thereby immobilizing the mammal.

The foregoing methods may be practiced utilizing a composition thatcomprises a botulinum toxin type A. In other embodiments of theinvention, the foregoing methods may be practiced with a compositionthat comprises botulinum toxin type B. In further embodiments of theinvention, the methods may be practiced with a composition thatcomprises a plurality of botulinum toxin serotypes, such as botulinumtoxin serotypes selected from the group consisting of botulinum toxinserotypes A, B, C₁, D, E, F and G. In certain embodiments of theinvention, purified botulinum toxins may be used. In other embodiments,modified botulinum toxins may be used. The compositions used in theforegoing methods may also include one or more amino acids in additionto the botulinum toxin and the polysaccharide.

In yet additional embodiments of the invention, the compositions used inthe foregoing methods can be administered intramuscularly to thepatient. In other embodiments, the compositions can be administeredsubcutaneously and/or intrathecally.

DESCRIPTION

The present invention is based upon the discovery that a Clostridialtoxin pharmaceutical composition, with a stabilized Clostridial toxin, areduced toxicity of the pharmaceutical composition and/or with a reducedantigenicity, as well as being free of animal derived proteins (such asa blood pooled or blood fraction derived human serum albumin or gelatin)can be made by using a polysaccharide as the primary or proteinstabilizer of the Clostridial toxin.

The present invention also encompasses a stable Clostridial toxincontaining pharmaceutical composition formulated free of any animalderived protein or donor pool albumin by incorporating both anpolysaccharide and an amino acid into the pharmaceutical composition. Inparticular, the present invention encompasses a stable botulinum toxincontaining pharmaceutical composition suitable for administration to apatient for therapeutic effects made by replacing the donor pool albuminpresent in known botulinum toxin containing pharmaceutical compositionswith a high molecular weight polysaccharide derived from starch and/orwith certain reactive amino acids.

Polysaccharide Containing Pharmaceutical Composition

I have discovered that a suitable replacement for albumin in aClostridial toxin pharmaceutical composition can be a compound which isneither another protein, nor a low molecular weight, non-proteincompound. Thus, I have discovered that particular high molecular weightpolysaccharides can function as neurotoxin stabilizers in apharmaceutical composition. As set forth below, an amino acid can also,or in the alternative, be added to the pharmaceutical composition toincrease the stability and useful storage life of the pharmaceuticalcomposition.

The polysaccharide used in the present invention can impart stability toa neurotoxin active ingredient, such as a botulinum toxin, present inthe pharmaceutical composition by: (1) reducing adhesion (commonlyreferred to as “stickiness”) of the botulinum toxin to surfaces, such asthe surfaces of laboratory glassware, vessels, the vial in which thepharmaceutical composition is reconstituted and the inside surface ofthe syringe used to inject the pharmaceutical composition. Adhesion ofthe botulinum toxin to surfaces can lead to loss of botulinum toxin andto denaturation of retained botulinum toxin, both of which reduce thetoxicity of the botulinum toxin present in the pharmaceuticalcomposition. (2) reducing the denaturation of the botulinum toxin and/ordissociation of the botulinum toxin from other non-toxin proteinspresent in the botulinum toxin complex, which denaturation and/ordissociation activities can occur because of the low dilution of thebotulinum toxin present in the pharmaceutical composition (i.e. prior tolyophilization or vacuum drying) and in the reconstituted pharmaceuticalcomposition. (3) reducing loss of botulinum toxin (i.e. due todenaturation or dissociation from non-toxin proteins in the complex)during the considerable pH and concentration changes which take placeduring preparation, processing and reconstitution of the pharmaceuticalcomposition.

The three types of botulinum toxin stabilizations provided by thepolysaccharide conserve and preserve the botulinum toxin with it nativetoxicity prior to injection of the pharmaceutical composition.

In certain embodiments of the invention, the pharmaceutical compositionsof the invention may comprise a plurality of botulinum toxin serotypes.In other words, the composition may include two or more differentbotulinum toxin serotypes. For example, a composition may includebotulinum toxin serotypes A and B. In another embodiment, a compositionmay include botulinum toxin serotypes A and E. Using a combination ofbotulinum toxin serotypes will permit caregivers to customize thecomposition to achieve a desired effect based on the condition beingtreated. In an additional embodiment of the invention, the compositionmay comprise a modified botulinum toxin. The modified botulinum toxinwill preferably inhibit the release of neurotransmitter from a neuron,but may have a greater or lower potency than the native botulinum toxin,or may have a greater or lower biological effect than the nativebotulinum toxin. Because the compositions of the invention may be usedfor relatively long-term treatment of animals, the compositions may beprovided in a relatively pure form. In one embodiment, the compositionsare of a pharmaceutical grade. In certain embodiments, the clostridialneurotoxin has a greater than 95% purity. In additional embodiments, theclostridial neurotoxin has a purity greater than 99%.

A preferred polysaccharide for use in the present composition comprisesa plurality of glucose monomers (mol wt 180) with one or moresubstituents on a majority of the glucose monomers, so that thepreferred polysaccharide has a molecular weight range of between about20 kD and about 800 kD. Surprisingly, such a polysaccharide canstabilize a neurotoxin component present in a pharmaceuticalcomposition. The present invention excludes from its scope disaccharideoligosaccharides with a weight average molecular weight of less thanabout 20 kD. The present invention also excludes from its scope cyclicpolymers such as the cyclodextrins. The latter two classes of compoundsare excluded from the scope of the present invention because the desiredstabilization characteristics of the preferred polysaccharide whilerequiring a relatively high molecular compound (i.e. molecular weight inexcess of 20 kD) do not require and indeed can make no use of the smalllipophilic cavity characteristic of the cyclodextrins, because thecyclodextrin lipophilic cavity is much smaller in size than the size ofthe neurotoxins stabilized by the preferred polysaccharides of thepresent invention. Additionally, the cyclodextrins are low molecularweight compounds comprising only about 6 to 8 glucose monomers.

The present invention also encompasses a method for stabilizingpharmaceutical compositions which contain a clostridial toxin with apolysaccharide. The stabilizing effect is achieved by bringing aclostridial toxin in contact with the polysaccharide. Examples ofsuitable polysaccharides within the scope of my invention includecertain starch and starch derivatives. As noted, the polysaccharideexhibits a stabilizing effect on the clostridial toxin. Furthermore, theeffect of the polysaccharide to stabilize a clostridial toxin can beenhanced by the addition of an amino acid.

Unexpectedly, I have discovered that 2-hydroxyethyl starch demonstratesa unique ability to stabilize the botulinum toxin present in a botulinumtoxin containing pharmaceutical composition, thereby providing apharmaceutical composition which is devoid of the potential forharboring a transmissible disease derived from human blood or bloodfraction donor pools or animal derived proteins like gelatin.

Thus, I have discovered that the particular high molecular weightpolysaccharide, hydroxyethyl starch, can stabilize the toxin duringformulation, drying, storage and reconstitution. Preferably, to furtherstabilize the protein active ingredient, an amino acid is also includedin the polysaccharide containing formulation.

The polysaccharide in the pharmaceutical composition is preferablyadmixed with the clostridial neurotoxin in an amount of about 1 μg ofpolysaccharide per unit of botulinum toxin to about 10 μg ofpolysaccharide per unit of botulinum toxin. More preferably thepolysaccharide in the pharmaceutical composition is admixed with theclostridial neurotoxin in an amount of about 4 μg of polysaccharide perunit of botulinum toxin to about 8 μg of polysaccharide per unit ofbotulinum toxin. In a most preferred embodiment, where thepolysaccharide is a hydroxyethyl starch, the hydroxyethyl starch in thepharmaceutical composition is preferably admixed with a botulinum toxintype A complex in an amount of about 5 μg of hydroxyethyl starch perunit of botulinum toxin to about 7 μg of hydroxyethyl starch per unit ofbotulinum toxin. Most preferably, the hydroxyethyl starch in thepharmaceutical composition is admixed with a botulinum toxin type Acomplex in an amount of about 6 μg of hydroxyethyl starch per unit ofbotulinum toxin. Since BOTOX® contains about 100 units of botulinumtoxin type A complex per vial and the average molecular weight ofhydroxyethyl starch is generally regarded as being between about 20 kDand about 2,500 kD, the most preferred concentration of hydroxyethylstarch is between about 1×10⁻⁹ moles per unit of botulinum toxin (M/U)to about 2×10⁻¹² moles per unit of botulinum toxin. In another preferredembodiment, for a 100 U botulinum toxin type A complex pharmaceuticalcomposition, about 600 μg of the hydroxyethyl starch and about 1 mg ofan amino acid, such as lysine, glycine, histidine or arginine isincluded in the formulation. Thus, my invention encompasses use of botha polysaccharide and an amino acid, or a polyamino acid to stabilize theneurotoxin active ingredient in the pharmaceutical composition.

Additionally, my invention also encompasses use of a suitable amino acidin a sufficient amount to stabilize the protein active ingredient in apharmaceutical composition, either in the presence of or to theexclusion of any polysaccharide being present in the formulation. Thus,I have surprisingly discovered that the inclusion of certain amino acidsinto a neurotoxin containing, pharmaceutical composition formulation canextend the useful shelf life of such a pharmaceutical composition. Thus,my invention encompasses a neurotoxin containing pharmaceuticalcomposition which includes an amino acid and the use of such apharmaceutical composition. Without wishing to be bound by theory, I canpostulate that since a neurotoxin, such as a botulinum toxin, issusceptible to oxidation, due to the presence of disulfide linkages inthe toxin complex, the inclusion of an oxidizable amino acid may act toreduce the probability that oxidizers, such as peroxides and freeradicals, will react with the neurotoxin. Thus, the likelihood that theoxidizable neurotoxin disulfide linkage will be oxidized by an oxidizer,such as peroxides and free radicals, can be reduced upon inclusion of anamino acid which can act as an oxidative sink, that is as a scavengerfor oxidizing compounds. A suitable amino acid is an amino acid which issubject to oxidation. Examples of preferred amino acids are methionine,cysteine, tryptophan and tyrosine. A particularly preferred amino acidis methionine.

A preferred embodiment of my invention can also include the use of twoor more amino acids either alone or in combination with a polysaccharideto stabilize the protein active ingredient in a pharmaceuticalcomposition. Thus, for a 100 U botulinum toxin type A containingpharmaceutical composition, about 0.5 mg of lysine and about 0.5 mg ofglycine can be used, either with or without between about 500 μg andabout 700 μg of hetastarch.

Thus, as set forth above, my invention encompasses a protein containing,pharmaceutical composition which includes a polysaccharide. Thepolysaccharide acts to stabilize the protein active ingredient in thepharmaceutical composition. Additionally, my invention also includes aprotein containing, pharmaceutical composition which includes apolysaccharide and an amino acid. Surprisingly, I have discovered thatthe inclusion of certain amino acids into a neurotoxin containing,pharmaceutical composition formulation which includes a carbohydrate canextend the useful shelf life of such a pharmaceutical composition. Thus,my invention encompasses a neurotoxin containing pharmaceuticalcomposition which includes both a polysaccharide and an amino acid andthe use of such a pharmaceutical composition. Furthermore, my inventionalso encompasses use of an amino acid without any polysaccharide beingpresent in the protein active ingredient pharmaceutical composition.

It is known that protein containing pharmaceutical compositions whichalso contain sugars, polysaccharides and/or carbohydrates (referred tohereafter as ‘reactive compounds”) are inherently unstable due to thefact that a protein and one of the three indicated reactive compoundscan undergo the well-described Maillard reaction. Extensive, largelyfruitless, research has been carried out to try and reduce the incidenceor prevalence of this (for example) protein-polysaccharide Maillardreaction, by reduction of moisture or by the use of non-reducing sugarsin the formulation. My discovery is based upon the observation thatinclusion of a high concentration of a highly reactive amino acidencourages the Maillard reaction to take place between the stabilizingpolysaccharide and the added amino acid. By providing an abundant aminesource for the carbohydrate to react with, the probability of theprotein drug (i.e. botulinum toxin active ingredient) becoming involvedin the Maillard is reduced, thereby reducing this degradation pathway ofthe protein active ingredient and in this manner thereby stabilizing theprotein active ingredient in the pharmaceutical composition.

Preferably, any compound containing a primary or secondary amine can beused for this purpose. Most preferred are amino acids, such as lysine,glycine, arginine. Polyamino acids, such as polylysine are alsosuitable. Cationic amino acids such as lysine may undergo ionicattraction, binding acidic proteins (e.g., botulinum toxins) and shieldthe active protein from contact with sugars. Polylysine, in addition tobeing larger and therefore more likely to act as a shield, provides theadditional advantage of being antibacterial.

Another aspect of my invention is to pre-react the sugar and amino acidcomponents to exhaust Maillard reaction potential before adding theactive protein component (botulinum toxin) to the sugar and amino acidformulation ingredients, thereby substantially limiting the activeprotein's exposure to Maillard reactions.

Thus, my invention encompasses a pharmaceutical composition containingand the use of an amino acids and polyamino acids as Maillard reactioninhibitors in protein (i.e. botulinum toxin) drug formulations whichcontain starches, sugars and/or polysaccharides.

The invention embodies formulations of active proteins (e.g., botulinumtoxin) in combination with a stabilizing starch, sugar, orpolysaccharide or combination of these, and an amino acid such aslysine.

Significantly, I have discovered that hydroxyethyl starch does notundergo or undergoes a much attenuated rate or level of Maillardreactions with a protein, such as a botulinum toxin, when hydroxyethylstarch is compared to other polysaccharides or carbohydrates.Additionally, I have discovered that inclusion of an amino acid enhancesthe preservation effect of hydroxyethyl starch, possibly by acting as acompetitive inhibitor, that is by competing with the toxin for Maillardreaction reactive sugars. For this purpose, amino acids such as lysine,glycine, arginine and histidine are preferred amino acids. Polyaminoacids, such as polylysine, which exhibit the desired competitiveinhibition behavior can also be used. Notably, the specified amino andpoly amino acids can also exhibit antimicrobial properties, providingtherefore the added benefit of reducing bacterial contamination in thepharmaceutical composition.

Reducing sugars, such as glucose and glucose polymers, undergo Maillardreaction with proteins. Even sugar alcohols like mannitol can react,albeit sometimes through contaminants or degradation products. Thereforea polysaccharide can stabilize the toxin for a period of time only tochemically react later, thereby causing reduced storage stability. It isobvious that the choice of polysaccharide is critical. I have discoveredthat the rate of hydroxyethyl starch participation in the Maillardreaction is very low. Additionally, I have found that hydroxyethylcellulose, although structurally very similar to hydroxyethyl starch, isunsuitable to use as a stabilizer, since I have found that hydroxyethylcellulose can rapidly react in a model system with lysine. This not onlymeans that hydroxyethyl starch has an obvious advantage over other sugar(i.e. polysaccharide) stabilizers, but that even excipients similar tohydroxyethyl starch, such as hydroxyethyl cellulose, can be unsuitableto use as stabilizers of a protein active ingredient in a pharmaceuticalformulation.

As noted, hydroxyethyl starch, can participate to at least some extent,in Maillard reactions. Thus, and as set forth above, a polysaccharidealone may not be sufficient to provide optimal stabilization of thetoxin. Thus, I discovered the advantages of inclusion of an amino acidto act as a competitive inhibitor. Without wishing to be bound bytheory, the hypothesis is that by providing another amine source, inhigh concentrations compared to the toxin, the probability of theMaillard reaction occurring with the toxin is reduced, therebystabilizing the toxin. Any amino acid can be used however lysine beinghighly reactive and is a preferred amino acid.

Significantly, I have discovered that a botulinum toxin pharmaceuticalcomposition formulated with hetastarch has a reduced toxicity, as wellas a reduced antigenicity. It can be hypothesized that the reducedtoxicity of the hetastarch botulinum toxin formulation upon eitherintramuscular or intravenous administration of amounts of botulinumtoxin which are toxic when the botulinum toxin is formulated with ahuman serum albumin, is due to hetastarch mediated sequestration of thebotulinum toxin by local and regional macrophages and monocytes of thereticuloendothelial system, where presumably the toxin undergoeslysosomal proteolysis. Thus, it is known that upon intravenousadministration of hetastarch as a plasma expander, the hetastarch can betaken up by macrophages and retained for prolonged periods in the liver,lung, spleen and skin. See e.g. Sirtl, C., et al., Tissue deposits ofhydroxyethyl starch (HES): dose-dependent and time-related, Br JAnaesth. 1999 April; 82(4):510-5; Reimann S., et al., [Hydroxyethylstarch accumulation in the skin with special reference to hydroxyethylstarch-associated pruritus]; Dtsch Med Wochenschr. 2000 Mar. 10;125(10):280-5; Parth E., et al., Histological and immunohistochemicalinvestigations of hydroxyethyl-starch deposits in rat tissues, Eur SurgRes. 1992; 24(1):13-21; Jurecka W., et al., Hydroxyethylstarch depositsin human skin—a model for pruritus?, Arch Dermatol Res. 1993;285(1-2):13-9

Thus, I have discovered that a botulinum toxin formulated withhetastarch not only retains the therapeutic paralytic activity of thetoxin at the injection site upon intramuscular or subcutaneous ofadministration of the formulation (i.e. the hetastarch present in theformulation does not interfere with the activity of the botulinum toxinat the neuromuscular junction) but that the hetastarch formulation alsohas a markedly reduced toxicity.

My invention also encompasses addition of a preservative, either in thediluent or formulation itself, to allow extended storage. A preferredpreservative is preserved saline containing benzyl alcohol.

A liquid formulation can be advantageous. A single-step presentation(e.g., pre-filled syringe) or a product configuration that the userperceives as a single-step presentation (e.g., dual-chambered syringe)would provide convenience by eliminating the reconstitution step.Freeze-drying is a complicated, expensive and difficult process. Liquidformulations are often easier and cheaper to produce. On the other handliquid formulations are dynamic systems and therefore more susceptibleto excipient interaction, fast reactions, bacterial growth, andoxidation than freeze-dried formulations. A compatible preservativemight be needed. Anti-oxidants such as methionine might also be usefulas scavengers especially if surfactants are used to reduce adsorption asmany of these compounds contain or produce peroxides. Any of thestabilizing excipients which can be used in a freeze-dried formulation(e.g., hydroxyethyl starch or an amino acid such, lysine) might beadapted to use in a liquid formulation to assist in reducing adsorptionand stabilize the toxin. Suspensions similar to those developed forinsulin are also good candidates. Additionally, stabilizing botulinumtoxin in a liquid vehicle might require a low pH vehicle as the toxin isreported to be labile above pH 7. This acidity could produce burning andstinging upon injection. A binary syringe could be employed. Inclusionof a co-dispensed buffer, sufficient to raise the pH to physiologiclevels, would alleviate injection discomfort of a low pH whilemaintaining the toxin at a low pH during storage. Another dual-chamberedsyringe option would include diluent and lyophilized material segregatedin separate chamber, only mixing upon use. This option provides theadvantages of a liquid formulation without the additional to resourcesand time.

Thus, the botulinum toxin can be prepared at low pH to be co-dispensedwith a buffer which raises the pH to at or near physiological pH at thetime of administration. The two chamber or binary syringe can have inthe first chamber (next to the plunger) a liquid formulation of abotulinum toxin with a pH between 3 to 6 (i.e. at pH 4.0). The secondchamber (next to the needle tip) can contain a suitable buffer, such asphosphate buffered saline at a higher pH (i.e. pH 7.0). Alternately, thefirst chamber can contain a saline diluent and the second chamber cancontain a freeze dried or lyophilized neurotoxin formulation. The twochambers can be joined in such a way and the buffering componentsselected in such a way that the solutions mix at or near the needle,thereby delivering the final solution at a physiological pH. Suitabletwo chamber syringes to use as pre-filled syringes for the purposes setforth herein can be obtained from Vetter Pharma-Fertigung of Yardley,Pa.

There are distinct advantages to formulating botulinum toxin at a lowpH. The toxin has a low isoelectric point (pI) and formulating proteinsnear their pI is a known way to stabilize a protein. Additionally, thetoxin is used at a very low concentration making surface adsorption aproblem. Use of a low pH solution can suppress ionization of toxin siteslikely to interact with surfaces. The syringe and plunger materials arematerials which reduce surface adsorption by the toxin. Suitable suchmaterials are polypropylene.

As discussed herein, the neurotoxin may be prepared and purified usingtechniques well-known in the art. The purified toxin may subsequently bediluted in a stabilizer such as a polysaccharide (e.g., hetastarch), ora recombinant serum albumin, or a serum albumin of the species of animalreceiving the neurotoxin. It is preferred that the stabilizer preventsor reduces denaturation of the toxin, and produces no, or minimal,immunogenic responses in the animal that will receive the toxin.Aliquots of the diluted toxin are then lyophilized using conventionalprocedures.

The lyophilized neurotoxin may be reconstituted before administering theneurotoxin to a subject by adding water, saline, or any buffer solutionto the lyophilized neurotoxin. In certain embodiments, sodium freebuffers may be preferred to help reduce denaturation of the neurotoxin.

The pharmaceutical compositions of the invention can be administeredusing conventional modes of administration. In preferred embodiments ofthe invention, the compositions are administered intramuscularly orsubcutaneously to the subject. In other embodiments, the compositions ofthe invention may be administered intrathecally. In addition, thecompositions of the invention may be administered with one or moreanalgesic or anesthetic agents.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the type, severity, andcourse of the condition being treated, the animal's health and responseto treatment, and the judgment of the treating doctor. Accordingly, themethods and dosages of the compositions should be tailored to theindividual subject.

By way of example, and not by way of limitation, it may be preferred toadminister the composition of the invention intramuscularly to reduce amuscle spasm.

My invention also encompasses a pharmaceutical composition comprising abotulinum toxin and a collagen for use to treat a variety of conditionswherein the botulinum toxin acts to paralyze a muscle and the collagenacts to provide a dermal filler.

As indicated above, dosages of the neurotoxin, such as botulinum toxin,in the compositions may vary. In one embodiment, the compositionscontain a therapeutically effective amount of neurotoxin, for example,between about 1 U and about 500 U of botulinum toxin type A. Preferablythe amounts are between about 10 U and about 300 U. More preferably theamount is between about 20 U and 250 U, such about 50 U to 200 U, or 70U.

Alternatively, botulinum toxin, such as botulinum toxin type A, can beadministered in amounts between about 10⁻³ U/kg and about 60 U/kg toalleviate pain experienced by a mammal. Preferably, the botulinum toxinused is administered in an amount of between about 10⁻² U/kg and about50 U/kg. More preferably, the botulinum toxin is administered in anamount of between about 10⁻¹ U/kg and about 40 U/kg. Most preferably,the botulinum toxin is administered in an amount of between about 1 U/kgand about 30 U/kg. In a particularly preferred embodiment of the presentdisclosed methods, the botulinum toxin is administered in an amount ofbetween about 1 U/kg and about 20 U/kg.

Compositions containing other serotypes of botulinum toxin may containdifferent dosages of the botulinum toxin. For example, botulinum toxintype B may be provided in a composition at a greater dose than acomposition containing botulinum toxin type A. In one embodiment of theinvention, botulinum toxin type B may be administered in an amountbetween about 1 U/kg and 150 U/kg. Botulinum toxin type B may also beadministered in amounts of up to 20,000 U (mouse units, as describedabove). In another embodiment of the invention, botulinum toxin types Eor F may be administered at concentrations between about 0.1 U/kg and150 U/kg. In addition, in compositions containing more than one type ofbotulinum toxin, each type of botulinum toxin can be provided in arelatively smaller dose than the dose typically used for a singlebotulinum toxin serotype. The combination of botulinum toxin serotypesmay then provide a suitable degree and duration of paralysis without anincrease in diffusion of the neurotoxins (e.g. see U.S. Pat. No.6,087,327).

EXAMPLES

The following examples set forth specific embodiments of the presentinvention and are not intended to limit the scope of the invention.

Example 1 Preparation and Potency of a Botulinum Toxin PharmaceuticalComposition Containing Human Serum Albumin (Prior Art) (Formulation I)

A botulinum toxin type A complex was obtained from a culture of the Hallstrain of Clostridium botulinum grown in a medium containing N-Z amineand yeast extract. The botulinum toxin type A complex was purified fromthe culture solution by a series of acid precipitations to a crystallinecomplex consisting of the active high molecular weight toxin protein andan associated hemagglutinin protein. The crystalline complex was thenre-dissolved in a solution containing saline and albumin and sterilefiltered (0.2 microns) prior to vacuum-drying. The vacuum driedcomposition was reconstituted with sterile, non-preserved saline priorto injection. Each vial of vacuum dried composition contains about 100units (U) of Clostridium botulinum toxin type A complex, 0.5 milligramsof human serum albumin and 0.9 milligrams of sodium chloride in asterile, vacuum-dried form without a preservative. This pharmaceuticalcomposition is Formulation I, sold commercially under the trade nameBOTOX® in 100 unit vials and is known to have an unchanged potency,after storage at −5 degrees Celsius for six months or longer followed byreconstitution in saline, of about 100 units.

Example 2 Preparation and Potency of a Botulinum Toxin PharmaceuticalComposition Containing 2-Hydroxyethyl Starch (Formulation II)

Botulinum toxin type A purified neurotoxin complex pharmaceuticalformulations were prepared in the same manner set forth in Example 1above, except that the 0.5 milligrams of albumin in Example 1 wasreplaced by either 500 μg or 600 μg of hetastarch. This pharmaceuticalcomposition is Formulation II (albumin free). It was determined thatfull potency was maintained upon preparation of the hetastarchcontaining formulations. Thus, Formulation II, with either 500 or 600 ugof hetastarch, had a potency as measured at the time of reconstitutionof the lyophilized, 100 U (±20 U) botulinum toxin type A Formulation IIof from 96 to 128 units. Three separate Formulation II batches hadpotency measurements at the time of reconstitution of, respectively,105, 111 and 128 units. Potency was measured using the standard mouseLD₅₀ assay.

Example 3 Preparation and Potency of Botulinum Toxin PharmaceuticalComposition Containing Hydroxyethyl Starch and Glycine (Formulation IIA)

Formulation IIA was made by adding 1 mg glycine USP, and 0.004 mg zincchloride USP to the Formulation II of Example 2, reconstituted withsodium chloride and it's potency was determined (T₀ potency) using themouse LD₅₀ assay. Lyophilized (i.e. not reconstituted) Formulation IIAwas then stored for seven months at −5° C. At the end of this sevenmonth period the potency of Formulation IIA was again determined uponreconstitution in sodium chloride, using the mouse LD₅₀ assay, to beessentially unchanged (i.e. potency differed by less than 5% from the T₀potency).

Example 4 Toxicity of a Botulinum Toxin Pharmaceutical CompositionContaining Human Serum Albumin Upon Intramuscular Administration(Formulation I)

An experiment was carried out to evaluate the toxicity of a botulinumtoxin pharmaceutical composition containing human serum albumin (i.e.BOTOX®) upon intramuscular administration.

Formulation I was prepared as in Example 1 (i.e. 100 units botulinumtoxin type A complex, 0.5 mg human serum albumin USP, 0.9 mg sodiumchloride USP, vacuum dried, without a preservative). The vacuum driedformulation was removed from the freezer, reconstituted with sodiumchloride and administered within four hours.

A Formulation I placebo containing 0.5 mg human serum albumin USP and0.9 mg sodium chloride USP was also prepared. The diluent used for bothformulation and placebo was 0.9% sodium chloride inj. USP.

Six separate lots of twelve Sprague Dawley albino rats (n=12, 6 male, 6female; 72 study rats plus 12 control rats for placebo injection], about10 weeks old, Charles River Labs, Hollister, Calif.) were injected withsingle intramuscular injections into the left gastrocnemius muscle ofthe left hind limb of Formulation I at doses of 5, 10, 50, 100, 200 or300 units/kg, respectively. A control group (n=12; 6 male, 6 female) wasinjected with single intramuscular injections of the vehicle (serumhuman and sodium chloride) at the equivalent dose volume (0.2 ml/kg).Rats were sacrificed and necropsied 14 days after the single injection.The parameters evaluated were mortality and morbundity, clinicalobservations, body weight, injected and non-injected gastrocnemiusmuscle weight, macroscopic observations and microscopic pathology of theinjected and non-injected gastrocnemius and biceps femoris muscles.

It was determined that intramuscular administration of Formulation Iinduced in rats local pharmacological effects (i.e. curling of the lefthind toes, limping, left abdominal distension, small left hind limb calfmuscles) at doses of 5 units/kg or higher, and clinical signs oftoxicity (i.e. chromodacryorrhea, chromorhinorrhea, piloerrection,hunched posture, soiled perianal region, immobility and morbundity) atdoses of 50 units/kg or higher. 50% of the rats died at the 100 units/kgdose level and 100% of the rats died at the 200 and 300 units/kg doselevels.

This experiment determined that Formulation I (a botulinum toxinpharmaceutical composition containing human serum albumin) can be lethalupon intramuscular administration to rats at a 100 unit/kg dose level.

Example 5 Reduced Toxicity of a Botulinum Toxin PharmaceuticalComposition Containing Hydroxyethyl Starch Upon IntramuscularAdministration

(Formulation II)

An experiment was carried out to evaluate the toxicity of a botulinumtoxin pharmaceutical composition containing 2-hydroxyethyl starch(Formulation II) upon intramuscular administration.

Formulation II was prepared as in Example 2 (i.e. 100 units botulinumtoxin type A complex, 0.6 mg hetastarch and 0.9 mg NaCl). The vacuumdried formulation was removed from the freezer, reconstituted withsodium chloride and administered within four hours.

A Formulation II placebo containing 0.6 mg hetastarch was also prepared.The diluent used for both formulation and placebo was 0.9% sodiumchloride inj. USP.

Four separate lots of 8 Sprague Dawley rats (n=8; 4 male, 4 female) (40rats total [including controls], 7-10 weeks old, Charles River Labs,Hollister, Calif.) were given single intramuscular injections into theleft gastrocnemius muscle of the left hind limb of Formulation II atdoses of 10, 50, 100 and 200 units/kg. A control group (n=8; 4 male, 4female) were injected with single intramuscular injections of thevehicle (serum human and sodium chloride) at the equivalent dose volume(0.2 mL/kg). Rats were sacrificed and necropsied 14 days after thesingle injection. The parameters evaluated were mortality, clinicalobservations, body weight, injected and non-injected gastrocnemiusmuscle weight, macroscopic observations.

Surprisingly no mortality was observed any dose level in any of the druginjected rats. Local pharmacological effects (curling of the left hindtoes, limping, decreased injected muscle weight) were observed in thedrug treated groups. Drug related clinical findings indicative oftoxicity included one incidence each of chromodacryorrhea andchromorrhinorrhea in one male rat in the 200 U/kg dose group.

As set forth in Example 4, intramuscular Formulation I induced in ratslocal pharmacological effects at doses of 5 units/kg or higher, clinicalsigns of toxicity at doses of 50 units/kg or higher and death at dosesof 100 units/kg or higher. However the experiment carried out in thisexample showed that intramuscular doses of Formulation II as high as 200units/kg induced only local pharmacological effects (i.e. curling of theleft hind toes, limping, palpably smaller left calf muscles anddecreased injected muscle weight), with minimal clinical signs oftoxicity and no mortality.

Hence, it can be concluded that Formulation II showed significantly lesstoxicity and similar local pharmacological effects, as compared toFormulation I.

Example 6 Reduced Toxicity of a Botulinum Toxin PharmaceuticalComposition Containing Hydroxyethyl Starch and Glycine UponIntramuscular Administration (Formulation IIA)

An experiment was carried out to evaluate the toxicity of a botulinumtoxin pharmaceutical composition containing 2-hydroxyethyl starch andglycine upon intramuscular administration.

Formulation IIA was prepared as in Example 3 (i.e. 100 units botulinumtoxin type A complex, 0.6 mg hetastarch and 0.9 mg NaCl) and 1 mgglycine USP, but no zinc chloride, was added to make the formulation.The vacuum dried formulation was removed from the freezer, reconstitutedwith sodium chloride and administered within four hours.

A Formulation IIA placebo containing 0.6 mg hetastarch and 1 mg glycinewas also prepared. The diluent used for both formulation and placebo was0.9% sodium chloride inj. USP.

Two separate lots of 5 female Sprague Dawley rats (n=5, all female) (15rats total [including controls], 7-10 weeks old, Charles River Labs,Hollister, Calif.) were given single intramuscular injections into theleft gastrocnemius muscle of the left hind limb of Formulation II atdoses of either 100 or 200 units/kg. A control group (n=5; all female)were injected with single intramuscular injections of the vehicle(hetastarch with glycine in sodium chloride diluent) at the equivalentdose volume (0.2 mL/kg). Rats were sacrificed and necropsied 14 daysafter the single injection. The parameters evaluated were mortality andmorbundity, clinical observations, body weight, injected andnon-injected gastrocnemius muscle weight, macroscopic observations andmicroscopic pathology of the injected and non-injected gastrocnemius andbiceps femoris muscles.

Surprisingly no mortality was observed at any dose level in any of thedrug injected rats. Nor were there observed any drug related clinicalsigns of toxicity at any dose level in any of the rats. Localpharmacological effects (curling of the left hind toes, limping,palpably smaller left calf muscles and decreased injected muscle weight)were observed in the drug treated groups.

As set forth in Example 4, intramuscular Formulation I induced in ratslocal pharmacological effects at doses of 5 units/kg or higher, clinicalsigns of toxicity at doses of 50 units/kg or higher and death at dosesof 100 units/kg or higher. However the experiment carried out in thisexample showed that intramuscular doses of Formulation IIA as high as200 units/kg induced only local pharmacological effects, with noclinical signs of toxicity or mortality. Hence, it can be concluded thata botulinum toxin pharmaceutical composition containing 2-hydroxyethylstarch (with or without glycine) has upon intramuscular administration asignificantly lower toxicity than does a botulinum toxin pharmaceuticalcomposition containing human serum albumin.

Since both Formulations II and IIA showed reduced toxicity (as comparedto Formulation I), but glycine was not present in Formulation II,therefore the reduced toxicity of Formulations II and IIA was due to thepresence of the hydroxyethyl starch present, and was not due to anyeffect of the glycine.

Example 7 Comparison of Toxicity of Botulinum Toxin PharmaceuticalCompositions Containing Human Serum Albumin or Hydroxyethyl Starch UponIntravenous Administration (Formulations I and IIA)

An experiment was carried out to evaluate the toxicity of a botulinumtoxin pharmaceutical composition containing 2-hydroxyethyl starch uponintravenous administration of the pharmaceutical composition. It wasexpected that an intravenously administered botulinum toxinpharmaceutical composition would result in widespread systemic effects,including muscle weakness, systemic toxicity, respiratory failure andsubject mortality.

Formulation I was prepared as set forth in Example 1 (i.e. 100 unitsbotulinum toxin type A complex, 0.5 mg human serum albumin (“HSA”) USP,0.9 mg sodium chloride USP). The vacuum dried formulation was removedfrom the freezer, reconstituted with sodium chloride and administeredwithin four hours.

Formulation IIA was prepared as in Example 3 (i.e. 100 units botulinumtoxin type A complex, 0.6 mg hetastarch and 0.9 mg NaCl) and 1 mgglycine USP, and 0.004 mg zinc chloride USP was added to theformulation. The vacuum dried formulation was removed from the freezer,reconstituted with sodium chloride and administered within four hours.

A Formulation I placebo containing 0.5 mg human serum albumin USP and0.9 mg sodium chloride USP was also prepared. A Formulation IIA placebocontaining 0.6 mg hetastarch, 1 mg glycine USP, and 0.004 mg zincchloride USP was also prepared.

The diluent used for both formulations and placebos was 0.9% sodiumchloride inj. USP.

Separate lots of 60 male and 60 female Sprague Dawley rats (n=12; sixmale, six female) (120 rats total, 6-10 weeks old, Charles River Labs,Hollister, Calif.) were injected with single intravenous injections ofthe two botulinum toxin formulations into the lateral tail vein at 10units per kg of animal weight, 50 units per kg, 100 units per kg or 200units per kg dose levels of the botulinum toxin type A. Two separatecontrol groups (n=12; six male, six female) were injected with singleintravenous injections of each of the two different placebos at anequivalent dose volume (0.2 ml/kg).

Surviving rats were sacrificed and necropsied 14 days after the singleinjection. The parameters evaluated were mortality, clinicalobservations, body weight and macroscopic observations.

The results of this experiment are shown by Table 1. Not unexpectedly,mortality occurred in all rats treated with ≧100 U/kg of any of theFormulation I. Surprisingly, no mortality was observed in any of therats treated with Formulation IIA, at doses as high as 200 U/kg. ThusFormulation IIA exhibited a significantly lower systemic toxicity ascompared to Formulation I.

TABLE 1 Toxicity of a Botulinum Toxin Hetastarch Formulation Compared toa Botulinum Toxin Human Serum Albumin Formulation Upon IntravenousAdministration Total No Toxin Dose Formulation or Dead/No. Percent GroupNo. (U/kg) Placebo Dosed Mortality 1 0 Placebo HSA 0/12 0 2 10 I 0/12 03 50 I 0/12 0 4 100 I 12/12  100 5 200 I 12/12  100 6 0 PlaceboHetastarch 0/12 0 7 10 IIA 0/12 0 8 50 IIA 0/12 0 9 100 IIA 0/12 0 10200 IIA 0/12 0

Example 8 Botulinum Toxin Pharmaceutical Composition Containing Lysine(Formulation IIC)

100 U botulinum toxin type A purified neurotoxin complex pharmaceuticalformulations are prepared in the same manner set forth in Example 1above, except that the 0.5 milligrams of albumin was replaced by 600 μgof hetastarch. In addition 1 mg of lysine is added to the formulation. Alyophilized, hetastarch plus lysine, albumin-free, 100 U botulinum toxintype A complex, pharmaceutical composition is then stored for one yearat −5° C. At the end of this one year period the potency of thishetastarch plus lysine, toxin formulation is determined, using the mouseadministration assay, to be essentially unchanged (i.e. potency differsby less than 5% from the original potency).

Example 9 Botulinum Toxin Pharmaceutical Composition ContainingHistidine (Formulation IID)

100 U botulinum toxin type A purified neurotoxin complex pharmaceuticalformulations are prepared in the same manner set forth in Example 1above, except that the 0.5 milligrams of albumin was replaced by 600 μgof hetastarch. In addition 1 mg of histidine is added to theformulation. A lyophilized, hetastarch plus histidine, albumin-free, 100U botulinum toxin type A complex, pharmaceutical composition is thenstored for one year at −5° C. At the end of this one year period thepotency of this hetastarch plus histidine, toxin formulation isdetermined, using the mouse administration assay, to be essentiallyunchanged (i.e. potency differs by less than 5% from the originalpotency).

Example 10 Botulinum Toxin Pharmaceutical Composition ContainingArginine (Formulation IIE)

100 U botulinum toxin type A purified neurotoxin complex pharmaceuticalformulations are prepared in the same manner set forth in Example 1above, except that the 0.5 milligrams of albumin was replaced by 600 μgof hetastarch. In addition 1 mg of arginine is added to the formulation.A lyophilized, hetastarch plus arginine, albumin-free, 100 U botulinumtoxin type A complex, pharmaceutical composition is then stored for oneyear at −5° C. At the end of this one year period the potency of thishetastarch plus arginine, toxin formulation is determined, using themouse administration assay, to be essentially unchanged (i.e. potencydiffers by less than 5% from the original potency).

Example 11 Botulinum Toxin Pharmaceutical Composition Containing AnAmino Acid

Botulinum toxin type A purified neurotoxin complex pharmaceuticalformulations can be prepared in the same manner set forth in Example 1above, except that the 0.5 milligrams of albumin can be replaced byabout 1 mg of an amino acid such as lysine, glycine, histidine orarginine. Thus a lyophilized, polysaccharide free, albumin free, glycinecontaining, botulinum toxin type A complex, pharmaceutical compositioncan be prepared and stored for at least one year −5° C., and at the endof this period can have a potency of which is essentially unchanged(i.e. potency can differ by less than 5% from the original potency).

Example 12 Use of a Botulinum Toxin Pharmaceutical Composition

A 48 year old male is diagnosed with a spastic muscle condition, such ascervical dystonia. Between about 10⁻³ U/kg and about 35 U/kg of abotulinum toxin type A pharmaceutical composition containing 600 μg ofhetastarch and 1 mg of an amino acid, such as lysine, is injectedintramuscularly into the patient. Within 1-7 days the symptoms of thespastic muscle condition are alleviated and alleviation of the symptomspersists for at least from about 2 months to about 6 months.

Example 13 Reduced Antigenicity Botulinum Toxin PharmaceuticalCompositions Containing Hydroxyethyl Starch Upon IntramuscularAdministration (Formulations I and IIA)

An experiment can be carried out to evaluate the antigenicity of abotulinum toxin pharmaceutical composition containing 2-hydroxyethylstarch, with and without glycine, upon intramuscular administration.

Formulations I, II and IIA can be prepared as set forth by Example 1-3,respectively. The vacuum dried formulations can be removed from thefreezer, reconstituted with sodium chloride and administered within fourhours. Formulations I, II and IIA placebos can be prepared as previouslyset forth.

Six separate lots of rabbits can be given periodic intramuscularinjections of Formulation I, Formulation II, Formulation IIA, or one ofthe three placebos (i.e. 3 control groups) over a six month period, at ahigh, but non-toxic dose level. It is expected that Formulations II andIIA will show a reduced generation of antibodies due to the reducedimmunogenicity of the carbohydrate hydroxyethyl starch of FormulationsII and IIA, as compared to the protein albumin present in Formulation I.

Example 14 Multiple Component Botulinum Toxin Formulations

As set forth briefly in the Definitions sections supra under thedefinition of “Pharmaceutical Composition” a multiple (i.e. two or more)component system for the making of a final formulation can provide thebenefit of allowing incorporation of ingredients which are notsufficiently compatible for long-term shelf storage with the firstcomponent of the two component system or which for other reasons it isnot desirable to include with the first component of the pharmaceuticalcomposition. In this manner what I refer to as adaptive neurotoxin (i.e.botulinum toxin) formulations can be prepared.

rHSA and HSA can require secondary stabilizers such asN-Acetyltryptophan, sodium caprylate, fatty acids, surfactants anddivalent cations for optimum long-term stability. Studies utilizingprobe formulations indicate that some of these secondary stabilizers mayinduce enhanced potency or stability on neurotoxin (i.e. botulinumtoxin) formulations. This example sets forth a way to add theappropriate concentration of these ingredients to obtain the desiredeffect. One way to accomplish this is to include the ingredient in thebase formulation. Another is to add it to a base formulation prior touse. As set forth herein, Zinc, for example, may enhance the potency orliquid stability when added to an existing neurotoxin (i.e. botulinumtoxin) formulation not containing zinc. Other beneficial stabilizingingredients can likewise be added in this manner.

A limitation of the current Botox® formulation is related to the usefullength of the product. Currently the recommended (for sterility reasons)life after reconstitution of Botox® is about four hours. This is due tothe fact that the product contains no preservative. The diluent, saline,also contains no preservative. Studies indicate that incorporation ofsome preservatives can degrade the toxin over a long storage time (i.e.up to 2 years) of the vacuum dried product. However, utilization of adiluent containing a preservative would expose the toxin to degradationprocesses for a much shorter time while still providing preservativeefficacy, that is the time of use, and thereby allow incorporation of apreservative.

A problem related to removal of protein stabilizers is clinicalperformance. Studies with hetastarch stabilized formulations indicatedifferences in performance (safety profile) possibly related todiffusion characteristics of the HES when compared to HSA. Otherstabilizers (such as povidone) which perform similarly to HSA have thusfar proven incompatible during storage, thereby limiting theirusefulness. Incorporating them into a diluent or reconstitution vehiclewould eliminate long-term exposure of the toxin to the incompatiblespecies during which such degradation could occur. Therefore a HES orsaline vacuum-dried formulation could be reconstituted in a vehiclecontaining a carrier, such as povidone, providing a shelf-stable productwith the desired clinical performance.

Buffer salts or physiological pH conditions also may cause degradationduring long-term storage. Enhanced storage stability may only beachievable by providing a suitable pH environment not suitable forinjection (burning/stinging), such as low pH. A two-part system wouldovercome these limitations. A low pH shelf-stable formulation could bediluted or reconstituted using a buffer to overwhelm the capacity of thefirst composition, thereby providing a comfortable physiologic pH forinjection.

Other advantages allow for changing the vehicle depending on use; i.e.,the carrier could be selected according to the desired clinicaldiffusion characteristics. For example, toxin vacuum-dried in SWI,reconstituted with HES for one indication (cosmetic) and HSA for another(therapeutic). Stabilizing the toxin in SWFI, then reconstituting withsaline (to obtain isotonicity) is another formulation option.

The final formulation might also be adapted to provide for patientsallergic to a particular ingredient (a collagen vehicle substituted fora patient allergic to gelatin, for example). Otherwise, the base toxinformulation could be reconstituted with different albumins derived froma particular species for use in veterinary applications (equine serumalbumin for use in horses, bovine serum albumin for use in cattle, asexamples).

The basis of the invention is a formulation combined with adaptive,specialized vehicles/diluents to obtain the desired characteristics. Theproduct can consist of three (or more) components; e.g., a vialcontaining a stable solid toxin and two pre-filled syringes containingdifferently formulated reconstitution vehicles.

Examples of three-component systems:

Version I (solid/liquid):

-   -   1. Toxin vacuum dried in NaCl    -   2. Reconstitution vehicle containing HSA or rHA    -   3. Second reconstitution vehicle containing HES

In this version of a pharmaceutical composition wherein the activeingredient is a botulinum toxin, reconstitution can be carried out so asto obtain a formulation wherein the diffusion profile of thepharmaceutical composition upon intramuscular injection is the same asor different from diffusion profile of a pharmaceutical compositionwherein the active ingredient is a botulinum toxin, but which containsalbumin instead of HES.

Version II (solid/liquid):

-   -   1. Toxin vacuum dried in NaCl    -   2. Reconstitution vehicle containing Povidone    -   3. Second reconstitution vehicle containing HES

In this version of a pharmaceutical composition (as in each of theadditional versions set forth below) wherein the active ingredient is abotulinum toxin, reconstitution can be carried out so as to obtain aformulation wherein the diffusion profile of the pharmaceuticalcomposition upon intramuscular injection is the same as or differentfrom diffusion profile of a pharmaceutical composition wherein theactive ingredient is a botulinum toxin, but which contains albumininstead of HES and povidone.

Version III (solid/liquid):

-   -   1. Toxin vacuum dried in SWFI (sterile water for injection)    -   2. Reconstitution vehicle containing Povidone    -   3. Second reconstitution vehicle containing HES (reconstitute        with Povidone to obtain Botox safety profile/HES to obtain        modulated safety profile)

Version IV (liquid/liquid):

-   -   1. Toxin liquid stabilized with a buffer at pH 4    -   2. Diluent liquid buffered to pH 7 containing HSA    -   3. Second diluent liquid buffered to pH 7 containing HES (dilute        with HSA diluent to obtain Botox safety profile/HES to obtain        modulated safety profile)

Version VI (solid/liquid):

-   -   1. Toxin vacuum dried in SWFI (sterile water for injection)    -   2. Reconstitution vehicle containing HSA or rHA and preservative    -   3. Second reconstitution vehicle containing HES and preservative        (reconstitute with HSA/rHA to obtain Botox safety profile/HES to        obtain modulated safety profile; preservative is compatible with        toxin to retain potency during specified time of use (e.g., 48        hours))

A pharmaceutical composition according to the invention disclosed hereinhas many advantages, including the following:

-   -   1. the pharmaceutical composition can be prepared free of any        blood product, such as albumin and therefore free of any blood        product infectious element such as a prion.    -   2. the pharmaceutical composition has stability and high %        recovery of toxin potency comparable to or superior to that        achieved with currently available pharmaceutical compositions.    -   3. reduced toxicity, as assessed by either intramuscular or        intravenous administration.    -   4. reduced antigenicity.

Various publications, patents and/or references have been cited herein,the contents of which, in their entireties, are incorporated herein byreference.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of stabilizing polysaccharides and aminoacids are within the scope of the present invention.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

1. A pharmaceutical composition comprising: (a) a therapeuticallyeffective amount of a botulinum toxin, and; (b) a polysaccharidecomprising a plurality of linked glucopyranose units, each glucopyranoseunit having a plurality of hydroxyl groups, wherein an average of about6 to about 9 of they hydroxyl groups present on each of theglucopyranoses present in the polysaccharide are substituted, through anether linkage, with a compound of the formulate (CH₂)_(n)—OH, where ncan be an integer from 1 to 4, wherein the pharmaceutical compositionhas a reduced toxicity.
 2. The pharmaceutical composition of claim 1,wherein the average molecular weight of a disaccharide unit of thepolysaccharide is between about 345 D and about 1,000 D.
 3. Thepharmaceutical composition of claim 1, wherein the polysaccharide is anethyl ether substituted polysaccharide.
 4. The pharmaceuticalcomposition of claim 1, wherein the botulinum toxin is selected from thegroup consisting of botulinum toxin types A, B, C, D, E, F and G.
 5. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is animal protein free.
 6. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition retains between about20% and about 100% of its potency for at least one year when stored at atemperature between −1° C. and about −15° C.
 7. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition is devoidof any albumin.
 8. The pharmaceutical composition of claim 1, whereinthe polysaccharide is a hydroxyethyl starch.
 9. The pharmaceuticalcomposition of claim 1, wherein the botulinum toxin is botulinum toxintype A.
 10. The composition of claim 1, wherein the polysaccharide isselected from the group consisting of hydroxymethyl starch, hydroxyethylstarch, hydroxypropyl starch, hydroxybutyl starch, and hydroxypentylstarch.
 11. A pharmaceutical composition comprising: (a) atherapeutically effective amount of a botulinum toxin type A, and; (b) apolysacoharide comprising a plurality of linked glucopyranose units,each glucopyranose unit having a plurality of hydroxyl groups, whereinan average of about 6 to about 9 of they hydroxyl groups present on eachof the glucopyranoses present in the polysaccharide are substituted,through an ether linkage, with a compound of the formulate (CH₂)_(n)—OH,where n can be an integer from 1 to 4, wherein the pharmaceuticalcomposition has a reduced toxicity.